The invention is related to insulin compositions with a high content of zinc atoms per six molecules of acylated insulin. The insulin is an acylated insulin and may be mixed with a further insulin analogue such as the rapid acting insulin Asp B28 human insulin.

1. A soluble pharmaceutical composition comprising an acylated insulin and further comprising more than 4 zinc atoms per 6 molecules of acylated insulin.

2. The pharmaceutical composition according to claim 1 comprising up to about 12 zinc atoms per 6 molecules of acylated insulin.

3. The pharmaceutical composition according to claim 1 comprising between about 4.3 and about 12 zinc atoms per 6 molecules of acylated insulin.

4. The pharmaceutical composition according to claim 1 comprising between about 4.5 and about 12 zinc atoms per 6 molecules of acylated insulin.

5. The pharmaceutical composition according to claim 1, wherein at least 85% of the acylated insulin is present as complexes which are acylated insulin dodecamers or complexes with a higher molecular weight than acylated insulin dodecamer.

6. The pharmaceutical composition according to claim 1, wherein the composition comprises a surfactant.

7. The pharmaceutical composition according to claim 1, wherein the insulin molecule has a side chain attached either to the α-amino group of the N-terminal amino acid residue of the B chain or to an Eε-amino group of a Lys residue present in the B chain of the parent insulin moiety via an amide bond, which side chain comprises at least one free carboxylic acid group or a group which is negatively charged at neutral pH, a fatty acid moiety with about 4 to about 32 carbon atoms in the carbon chain; and possible one or more linkers linking the individual components in the side chain together via amide bonds.

8. The pharmaceutical composition according to claim 1, wherein the side chain comprises at least one aromatic group.

9. The pharmaceutical composition according to claim 1, wherein the side chain comprises at least one difunctional PEG group.

10. The pharmaceutical composition according to claim 1, wherein the insulin molecule has a side chain attached to the Eε-amino group of a Lys residue present in the B chain of the parent insulin, the side chain being of the general formula: —W—X—Y-Z2 wherein W is: an α-amino acid residue having a carboxylic acid group in the side chain which residue forms, with one of its carboxylic acid groups, an amide group together with ε-amino group of a Lys residue present in the B chain of the parent insulin; a chain composed of two, three or four α-amino acid residues linked together via amide carbonyl bonds, which chain—via an amide bond—is linked to an ε-amino group of a Lys residue present in the B chain of the parent insulin, the amino acid residues of W being selected from the group of amino acid residues having a neutral side chain and amino acid residues having a carboxylic acid group in the side chain so that W has at least one amino acid residue which has a carboxylic acid group in the side chain; or a covalent bond from X to an ε-amino group of a Lys residue present in the B chain of the parent insulin; X is: —CO—; —CH(COOH)CO—; —CO—N(CH2COOH)CH2CO—; —CO—N(CH2COOH)CH2CON(CH2COOH)CH2CO—; —CO—N(CH2CH2COOH)CH2CH2CO—; —CO—N(CH2CH2COOH)CH2CH2CON(CH2CH2COOH)CH2CH2CO—; —CO—NHCH(COOH)(CH2)4NHCO—; —CO—N(CH2CH2COOH)CH2CO—; or —CO—N(CH2COOH)CH2CH2CO—. that a) when W is an amino acid residue or a chain of amino acid residues, via a bond from the underscored carbon forms an amide bond with an amino group in W, or b) when W is a covalent bond, via a bond from the underscored carbonyl carbon forms an amide bond with an ε-amino group of a Lys residue present in the B chain of the parent insulin; Y is: —(CH2)m— where m is an integer in the range of 6 to 32; a divalent hydrocarbon chain comprising 1, 2 or 3 —CH═CH— groups and a number of —CH2— groups sufficient to give a total number of carbon atoms in the chain in the range of 10 to 32; and Z2 is: —COOH; —CO-Asp; —CO-Glu; —CO-Gly; —CO-Sar; —CH(COOH)2; —N(CH2COOH)2; —SO3H; or —PO3H and any Zn2+ complexes thereof, provided that when W is a covalent bond and X is —CO—, then Z is different from —COOH.

11. The pharmaceutical composition according to claim 1, wherein the acylated insulin is having a formula wherein Ins is the parent insulin moiety which via the α-amino group of the N-terminal amino acid residue of the B chain or an ε-amino group of a Lys residue present in the B chain of the insulin moiety is bound to the CO— group in the side chain via an amide bond; X4 is —(CH2)n where n is 1, 2, 3, 4, 5 or 6; NR, where R is hydrogen or —(CH2)p—COOH; —(CH2)p—SO3H; —(CH2)p—PO3H2; —(CH2)p—O—SO3H2; —(CH2)p—O—PO3H2; arylene substituted with 1 or 2 —(CH2)p—O—COOH groups; —(CH2)p-tetrazolyl, where p is an integer in the range of 1 to 6; —(CR1R2)q—NR—CO—, where R1 and R2 independently of each other and independently for each value of q can be H, —COOH, or OH, q is 1-6 and R is defined as above; —((CR3R4)q1—NR—CO)2-4—, where R3 and R4 independently of each other and independently for each value of q1 can be H, —COOH, or OH, q1 is 1-6 and R is defined as above; or a bond W1 is arylene or heteroarylene, which may be substituted with one or two groups selected from the group consisting of —COOH, —SO3H, and —PO3H2 and tetrazolyl, or W1 is a bond; m is 0, 1, 2, 3, 4, 5 or 6; X5 is where R is defined as above; or a bond; Y1 is —(CR1R2)q—NR—CO—, where R1 and R2 independently of each other and independently for each value of q can be H, —COOH, a bond or OH, q is 1-6; and R is defined as above; NR where R is defined as above; —((CR3R4)q1—NR—CO)2-4—, where R3 and R4 independently of each other and independently for each value of q1 can be H, —COOH, or OH, q1 is 1-6 and R is defined as above; or a bond; Q7 is —(CH2)r— where r is an integer from 4 to 22; a divalent hydrocarbon chain comprising 1, 2 or 3 —CH═CH— groups and a number of —CH2— groups sufficient to give a total number of carbon atoms in the chain in the range of 4 to 22; or a divalent hydrocarbon chain of the formula —(CH2)s-Q8-(C6H4)v1-Q9-(CH2)W-Q10-(C6H4)v2-Q11-(CH2)t-Q12-(C6H4)v3-Q13-(CH2)z— wherein Q8-Q13 independently of each other can be O; S or a bond; where s, w, t and z independently of each other are zero or an integer from 1 to 10 so that the sum of s, w, t and z is in the range from 4 to 22, and v1, v2, and v3 independently of each other can be zero or 1, provided that when W1 is a bond then Q7 is not a divalent hydrocarbon chain of the formula —(CH2)v4C6H4(CH2)W1— wherein v4 and w1 are integers or one of them is zero so that the sum of v4 and w1 is in the range of 6 to 22; and Z1 is: —COOH; —CO-Asp; —CO-Glu; —CO-Gly; —CO-Sar; —CH(COOH)2; —N(CH2COOH)2; —SO3H —PO3H2; —O—SO3H; —O—PO3H2; -tetrazolyl or —O—W2, where W2 is arylene or heteroarylene substituted with one or two groups selected from —COOH, —SO3H, and —PO3H2 and tetrazolyl; provided that if W1 is a bond and v1, v2 and v3 are all zero and Q8-13 are all a bonds, then Z1 is O—W2 and any Zn2+ complex thereof.

12. The pharmaceutical composition to claim 1, wherein the acylated insulin is having a formula wherein Ins is the parent insulin moiety which via the α-amino group of the N-terminal amino acid residue of the B chain or an ε-amino group of a Lys residue present in the B chain of the insulin moiety is bound to the CO— group in the side chain via an amide bond; each n is independently 0, 1, 2, 3, 4, 5 or 6; Q1, Q2, Q3, and Q4 independently of each other can be (CH2CH2O)s—; (CH2CH2CH2O)s—; (CH2CH2CH2CH2O)s—; (CH2CH2OCH2CH2CH2CH2O)s— or (CH2CH2CH2OCH2CH2CH2CH2O)s— where s is 1-20 —(CH2)r— where r is an integer from 4 to 22; or a divalent hydrocarbon chain comprising 1, 2 or 3 —CH═CH— groups and a number of —CH2— groups sufficient to give a total number of carbon atoms in the chain in the range of 4 to 22; —(CH2)t— or —(CH2OCH2)t—, where t is an integer from 1 to 6; —(CR1R2)q—, where R1 and R2 independently of each other can be H, —COOH, (CH2)1-6COOH and R1 and R2 can be different at each carbon, and q is 1-6, —((CR3R4)q1)1—(NHCO—(CR3R4)q1—NHCO)1-2—((CR3R4)q1)1 or ((CR3R4)q1)1—(CONH—(CR3R4)q1—CONH)1-2—((CR3R4)q1—)—, —((CR3R4)q1)1—(NHCO—(CR3R4)q1—CONH)1-2—((CR3R4)q1)1 or —((CR3R4)q1)1—(CONH—(CR3R4)q1—NHCO)1-2—((CR3R4)q1)1 where R3 and R4 independently of each other can be H, —COOH, and R3 and R4 can be different at each carbon, and q1 is 1-6-, or a bond; with the proviso that Q1-Q4 are different; X1, X2 and X3 are independently O; a bond; or where R is hydrogen or —(CH2)p—COOH, —(CH2)p—SO3H, —(CH2)p—PO3H2, —(CH2)p—O—SO3H; —(CH2)p—O—PO3H2; or —(CH2)p-tetrazol-5-yl, where each p independently of the other p's is an integer in the range of 1 to 6; and Z is: —COOH; —CO-Asp; —CO-Glu; —CO-Gly; —CO-Sar; —CH(COOH)2; —N(CH2COOH)2; —SO3H —OSO3H —OPO3H2 —PO3H2 or -tetrazol-5-yl and any Zn2+ complex thereof.

13. The pharmaceutical composition according to claim 1, wherein the parent insulin is a desB30 human insulin analogue.

14. The pharmaceutical composition according to claim 1 further comprising a rapid acting insulin

15. The pharmaceutical composition according to claim 1, wherein at least 85% of the rapid acting insulin is present as rapid acting insulin hexamer or complexes with a smaller molecular weight than rapid acting insulin hexamers.

17. A method for producing a pharmaceutical composition comprising an acylated insulin wherein more than about 4 zinc atoms per 6 molecules of acylated insulin are added to the composition.

18. A method according to claim 17 wherein up to about 12 zinc atoms per 6 molecules of acylated insulin are added to the composition.

19. A method according to claim 17 wherein between about 4.3 and about 12 zinc atoms per 6 molecules of acylated insulin are added to the composition.

20. A method according to claim 17 wherein the zinc is added to the composition before addition of a preservative.

21. A method according to claim 17 wherein the zinc is added to the composition after addition of a preservative.

22. A method according to claim 17, wherein part of the zinc is added before addition of a preservative and part of the zinc is added after addition of a preservative

23. A method according to claim 17, wherein the preservative is phenol and/or m-cresol.

24. A method according to claim 17, wherein a surfactant is mixed with the pharmaceutical composition.

25. A method according to claim 17, wherein acylated insulin has a side chain attached to the ε-amino group of a Lys residue present in the B chain of the parent insulin, the side chain being of the general formula: —W—X—Y-Z2 wherein W is: an α-amino acid residue having a carboxylic acid group in the side chain which residue forms, with one of its carboxylic acid groups, an amide group together with ε-amino group of a Lys residue present in the B chain of the parent insulin; a chain composed of two, three or four α-amino acid residues linked together via amide carbonyl bonds, which chain—via an amide bond—is linked to an ε-amino group of a Lys residue present in the B chain of the parent insulin, the amino acid residues of W being selected from the group of amino acid residues having a neutral side chain and amino acid residues having a carboxylic acid group in the side chain so that W has at least one amino acid residue which has a carboxylic acid group in the side chain; or a covalent bond from X to an ε-amino group of a Lys residue present in the B chain of the parent insulin; X is: —CO—; —CH(COOH)CO—; —CO—N(CH2COOH)CH2CO—; —CO—N(CH2COOH)CH2CON(CH2COOH)CH2CO—; —CO—N(CH2CH2COOH)CH2CH2CO—; —CO—N(CH2CH2COOH)CH2CH2CON(CH2CH2COOH)CH2CH2CO—; —CO—NHCH(COOH)(CH2)4NHCO—; —CO—N(CH2CH2COOH)CH2CO—; or —CO—N(CH2COOH)CH2CH2CO—. that a) when W is an amino acid residue or a chain of amino acid residues, via a bond from the underscored carbon forms an amide bond with an amino group in W, or b) when W is a covalent bond, via a bond from the underscored carbonyl carbon forms an amide bond with an ε-amino group of a Lys residue present in the B chain of the parent insulin; Y is: —(CH2)m— where m is an integer in the range of 6 to 32; a divalent hydrocarbon chain comprising 1, 2 or 3 —CH═CH— groups and a number of —CH2— groups sufficient to give a total number of carbon atoms in the chain in the range of 10 to 32; and Z2 is: —COOH; —CO-Asp; —CO-Glu; —CO-Gly; —CO-Sar; —CH(COOH)2; —N(CH2COOH)2; —SO3H; or —PO3H and any Zn2+ complexes thereof, provided that when W is a covalent bond and X is —CO—, then Z is different from —COOH.

26. A method according to claim 17, wherein the acylated insulin is having a formula wherein Ins is the parent insulin moiety which via the α-amino group of the N-terminal amino acid residue of the B chain or an ε-amino group of a Lys residue present in the B chain of the insulin moiety is bound to the CO— group in the side chain via an amide bond; X4 is —(CH2)n where n is 1, 2, 3, 4, 5 or 6; NR, where R is hydrogen or —(CH2)p—COOH; —(CH2)p—SO3H; —(CH2)p—PO3H2; —(CH2)p—O—SO3H2; —(CH2)p—O—PO3H2; arylene substituted with 1 or 2 —(CH2)p—O—COOH groups; —(CH2)p-tetrazolyl, where p is an integer in the range of 1 to 6; —(CR1R2)q—NR—CO—, where R1 and R4 independently of each other and independently for each value of q can be H, —COOH, or OH, q is 1-6 and R is defined as above; —((CR3R4)q1—NR—CO)2-4—, where R3 and R4 independently of each other and independently for each value of q1 can be H, —COOH, or OH, q1 is 1-6 and R is defined as above; or a bond W1 is arylene or heteroarylene, which may be substituted with one or two groups selected from the group consisting of —COOH, —SO3H, and —PO3H2 and tetrazolyl, or W1 is a bond; m is 0, 1, 2, 3, 4, 5 or 6; X5 is where R is defined as above; or a bond; Y1 is —(CR1R2)q—NR—CO—, where R1 and R2 independently of each other and independently for each value of q can be H, —COOH, a bond or OH, q is 1-6; and R is defined as above; NR where R is defined as above; —((CR3R4)q1—NR—CO)2-4—, where R3 and R4 independently of each other and independently for each value of q1 can be H, —COOH, or OH, q1 is 1-6 and R is defined as above; or a bond; Q7 is —(CH2)r— where r is an integer from 4 to 22; a divalent hydrocarbon chain comprising 1, 2 or 3 —CH═CH— groups and a number of —CH2— groups sufficient to give a total number of carbon atoms in the chain in the range of 4 to 22; or a divalent hydrocarbon chain of the formula —(CH2)s-Q8-(C6H4)v1-Q9-(CH2)W-Q10-(C6H4)v2-Q11-(CH2)t-Q12-(C6H4)v3-Q13-(CH2)z— wherein Q8-Q13 independently of each other can be O; S or a bond; where s, w, t and z independently of each other are zero or an integer from 1 to 10 so that the sum of s, w, t and z is in the range from 4 to 22, and v1, v2, and v3 independently of each other can be zero or 1, provided that when W1 is a bond then Q7 is not a divalent hydrocarbon chain of the formula —(CH2)v4C6H4(CH2)W1— wherein v4 and w1 are integers or one of them is zero so that the sum of v4 and w1 is in the range of 6 to 22; and Z1 is: —COOH; —CO-Asp; —CO-Glu; —CO-Gly; —CO-Sar; —CH(COOH)2; —N(CH2COOH)2; —SO3H —PO3H2; O—SO3H; O—PO3H2; -tetrazolyl or —O—W2, where W2 is arylene or heteroarylene substituted with one or two groups selected from —COOH, —SO3H, and —PO3H2 and tetrazolyl; provided that if W1 is a bond and v1, v2 and v3 are all zero and Q8-13 are all a bonds, then Z1 is O—W2 and any Zn2+ complex thereof.

27. A method according to claim 17, wherein the acylated insulin is having a formula wherein Ins is the parent insulin moiety which via the α-amino group of the N-terminal amino acid residue of the B chain or an ε-amino group of a Lys residue present in the B chain of the insulin moiety is bound to the CO— group in the side chain via an amide bond; each n is independently 0, 1, 2, 3, 4, 5 or 6; Q1, Q2, Q3, and Q4 independently of each other can be (CH2CH2O)s—; (CH2CH2CH2O)s—; (CH2CH2CH2CH2O)s—; (CH2CH2OCH2CH2CH2CH2O)s— or (CH2CH2CH2OCH2CH2CH2CH2O)s— where s is 1-20 —(CH2)r— where r is an integer from 4 to 22; or a divalent hydrocarbon chain comprising 1, 2 or 3 —CH═CH— groups and a number of —CH2— groups sufficient to give a total number of carbon atoms in the chain in the range of 4 to 22; —(CH2)t— or —(CH2OCH2)t—, where t is an integer from 1 to 6; —(CR1R2)q—, where R1 and R2 independently of each other can be H, —COOH, (CH2)16COOH and R1 and R2 can be different at each carbon, and q is 1-6, —((CR3R4)q1)1—(NHCO—(CR3R4)q1—NHCO)1-2—((CR3R4)q1)1 or —((CR3R4)q1)1—(CONH—(CR3R4)q1—CONH)1-2—((CR3R4)q1—)—, —((CR3R4)q1)1—(NHCO—(CR3R4)q1—CONH)1-2—((CR3R4)q1)1 or —((CR3R4)q1)1—(CONH—(CR3R4)q1—NHCO)1-2—((CR3R4)q1)1 where R3 and R4 independently of each other can be H, —COOH, and R3 and R4 can be different at each carbon, and q1 is 1-6-, or a bond; with the proviso that Q1-Q4 are different; X1, X2 and X3 are independently O; a bond; or where R is hydrogen or —(CH2)p—COOH, —(CH2)p—SO3H, —(CH2)p—PO3H2, —(CH2)p—O—SO3H; —(CH2)p—O—PO3H2; or —(CH2)p-tetrazol-5-yl, where each p independently of the other p's is an integer in the range of 1 to 6; and Z is: —COOH; —CO-Asp; —CO-Glu; —CO-Gly; —CO-Sar; —CH(COOH)2; —N(CH2COOH)2; —SO3H —OSO3H —OPO3H2 —PO3H2 or -tetrazol-5-yl and any Zn2+ complex thereof.

28. A method according to claim 17, wherein the parent insulin is a desB30 human insulin analogue.

29. A method according to claim 17, wherein a rapid acting insulin is mixed with the composition.

32. A pharmaceutical composition for the treatment of diabetes in a patient in need of such treatment, comprising a therapeutically effective amount of a pharmaceutical composition according to claim 1 together with a pharmaceutically acceptable carrier.

33. A method of treating diabetes in a patient in need of such a treatment, comprising administering to the patient a therapeutically effective amount of a pharmaceutical composition according to claim 1 together with a pharmaceutically acceptable carrier.

34. A method according to claim 33 for pulmonary treatment of diabetes.

35. (canceled)

36. (canceled)

Description:

FIELD OF THE INVENTION

The present invention relates to pharmaceutical compositions of acylated insulin with a prolonged action profile and high zinc content. Further the invention relates to a method for producing a composition with a prolonged action profile and high zinc content and a method for manufacturing a composition for the treatment of diabetes.

BACKGROUND OF THE INVENTION

Currently, the treatment of diabetes, both type 1 diabetes and type 2 diabetes, relies to an increasing extent on the so-called intensive insulin treatment. According to this regimen, the patients are treated with multiple daily insulin injections comprising one or two daily injections of a long acting insulin to cover the basal insulin requirement supplemented by bolus injections of a rapid acting insulin to cover the insulin requirement related to meals.

Long acting insulin compositions are well known in the art. Thus, one main type of long acting insulin compositions comprises injectable aqueous suspensions of insulin crystals or amorphous insulin. In these compositions, the insulin compounds utilized typically are protamine insulin, zinc insulin or protamine zinc insulin.

Certain drawbacks are associated with the use of insulin suspensions. Thus, in order to secure an accurate dosing, the insulin particles must be suspended homogeneously by gentle shaking before a defined volume of the suspension is withdrawn from a vial or expelled from a cartridge. Also, for the storage of insulin suspensions, the temperature must be kept within more narrow limits than for insulin solutions in order to avoid lump formation or coagulation.

While it was earlier believed that protamines were non-immunogenic, it has now turned out that protamine insulin crystals can be immunogenic in man and that their use for medical purposes may lead to formation of antibodies. Also, evidence has been found that the protamine crystal is itself immunogenic. Therefore, with some patients the use of long acting insulin compositions containing protamines must be avoided.

Another type of long acting insulin compositions are solutions having a pH value below physiological pH from which the insulin will precipitate because of the rise in the pH value when the solution is injected. A drawback with these solutions is that the particle size distribution of the precipitate formed in the tissue on injection, and thus the release profile of the medication, depends on the blood flow at the injection site and other parameters in a somewhat unpredictable manner. A further drawback is that the solid particles of the insulin may act as a local irritant causing inflammation of the tissue at the site of injection.

Insulin is a 51 amino acid peptide hormone produced in the islets of Langerhans in the pancreas. Its primary function, acting as a monomer, is to facilitate the transport of glucose molecules across the cell membranes of adipose and muscle tissue by binding to and activating a transmembrane receptor.

A distinctive property of insulin is its ability to associate into hexamers, in which form the hormone is protected from chemical and physical degradation during biosynthesis and storage. X-ray crystallographic studies on insulin show that the hexamer consists of three dimers related by a 3-fold axis of rotation. These dimers are closely associated through the interaction of two zinc ions at its core positioned on the 3-fold axis.

When human insulin is injected into the subcutis in the form of a high-concentration pharmaceutical formulation it is self associated, and here dissociation into monomers is relatively slow. Hexamers and dimers of insulin are slower to penetrate capillary wall than monomers.

Zinc and phenolic additives are regularly used in therapeutic insulin preparations to promote hexamer formation as a precaution against degradation during storage. In this form, however, the action of injected insulin is delayed while the hexamers diffuse through the subcutis and dissociate into dimers and monomers.

Formulations of insulin are usually prepared by dissolving insulin in a small volume of water under acidic conditions. Zinc is then added to the formulation followed by a neutralisation and addition of preservatives like phenol and m-cresol. The pharmaceutical formulation of these insulins are given as about 2, 3 or 4 zinc atoms per hexamer insulin.

WO 2005/012347 discloses another group of acylated insulin derivatives comprising additional negatively charge compared to the acylated insulins disclosed in WO 95/07931. The pharmaceutical formulation of these acylated insulins are given as 2, 3 or 4 zinc atoms per hexamer insulin.

WO 2003/094956 disclose stable insulin formulations prepared by mixing a monomeric insulin and a soluble acylated insulin analogue. The formulations contains from about 2.3 to about 4.5 Zn2+ per hexamer insulin. The acylated insulin analogue according to this invention is insulin detemir.

From WO 2003/094951 it is known to formulate a stable soluble insulin formulation having both fast and long action. The formulation has a content of zinc in the range from about 2.3 to about 4.5 Zn2+ per hexamer insulin. The acylated insulin analogue according to this invention is insulin detemir.

Human insulin has three primary amino groups: the N-terminal group of the A-chain and of the B-chain and the ε-amino group of LysB29. Several insulin derivatives which are substituted in one or more of these groups are known in the prior art. Thus, U.S. Pat. No. 3,528,960 (Eli Lilly) relates to N-carboxyaroyl insulins in which one, two or three primary amino groups of the insulin molecule has a carboxyaroyl group.

EP 894095 discloses insulin derivatives wherein the N-terminal group of the B-chain and/or the ε-amino group of Lys in position B28, B29 or B30 has a substituent of the formula —CO—W—COOH where W can be a long chain hydrocarbon group. These insulin derivatives have a prolonged profile of action and are soluble at physiological pH values.

WO 95/07931 discloses protracted insulin derivatives wherein a liphophilic side chain is attached either to the α-amino group of the N-terminal amino acid residue of the B chain or to the ε-amino group of a Lys residue present in the B chain of the parent insulin.

The mechanism behind slow absorption of insulin detemir has been studied by Havelund et al. (The mechanism of protraction of insulin detemir, a long-acting acylated analog of human insulin, Pharmaceutical Research, 21 (2004)1498-1504). Insulin formulations are prepared by adding 2 zinc atoms per hexamer insulin followed by glycerol, phenol, m-cresol and sodium phosphate.

In Whittingham et al (Crystallographic and solution studies of N-Lithocholyl insulin: a new generation of prolonged-acting human insulin, Biochemistry 2004, 43, 5987-5995) a formulation of acylated insulin analogue is prepared. The insulin formulation is prepared by adding 2-2.5 zinc atoms per hexamer insulin followed by glycerol and phenol. The structures of the insulin is measured by size exclusion chromatography.

The present invention is related to certain pharmaceutical compositions of acylated insulins which solves the problems of the prior art.

SUMMARY OF THE INVENTION

The present invention is related to a soluble pharmaceutical composition comprising an acylated insulin comprising more than 4 zinc atoms per 6 molecules of the acylated insulin.

The zinc content may be up to about 12 zinc atoms per 6 molecules of acylated insulin. The upper limit for the zinc content is the content of zinc which would cause precipitation of the insulin and turning the solution into a suspension.

In one aspect of the invention the pharmaceutical composition comprises between about 4.3 and about 12 zinc atoms per 6 molecules of acylated insulin or between about 4.5 and about 12 zinc atoms per 6 molecules of acylated insulin. In a further aspect of the invention the pharmaceutical composition comprises between about 5 and about 11.4 zinc atoms per 6 molecules of acylated insulin or between about 5.5 and about 10 zinc atoms per 6 molecules of acylated insulin. In a further aspect the pharmaceutical composition comprises between about 6 and about 10.5 zinc atoms per 6 molecules of acylated insulin or the pharmaceutical composition comprises between about 6.5 and about 10 zinc atoms per 6 molecules of acylated insulin or the pharmaceutical composition comprises between about 7 and about 9 zinc atoms per 6 molecules of acylated insulin. In a further aspect the pharmaceutical composition comprises citrate from about one third to about 3 times the zinc concentration.

The insulin molecules of the present invention associate with each other to form complexes comprising zinc. These insulin-zinc complexes can be present in the pharmaceutical formulation as hexamers, dodecamers or complexes with a higher molecular weight than dodecamers. All kinds of insulin form complexes with zinc, eg. Human insulin, acylated insulin (insulin derivatives) and insulin analogues.

In one aspect of the invention at least 85% of the acylated insulin is present as complexes which are acylated insulin dodecamers or complexes with a higher molecular weight than acylated insulin dodecamer. In one aspect of the invention at least 90, 92, 95, 96, 97, 98, 99 or 99.5% of the acylated insulin is present as complexes which are acylated insulin dodecamers or complexes with a higher molecular weight than acylated insulin dodecamer. In one aspect of the invention, the pharmaceutical composition comprises a surfactant. The surfactant can be present in an amount of 0.0005-0.01% based on the weight of the pharmaceutical composition. In one aspect the surfactant can be present in an amount of 0.0005-0.007% based on the weight of the composition. An example of a surfactant could be polysorbate 20, which can be present in the composition in an amount of 0.001-0.003% based on the weight of the composition. Another example is poloxamer 188, which can be present in an amount of 0.002-0.006% based on the weight of the composition.

The insulin molecule may be acylated at various positions in the insulin molecule. In one aspect the insulin is acylated in the F-amino group of a Lys residue in a position in the B-chain of the parent insulin molecule in particularly in the ε-amino group of the B29 lysine group in the human insulin molecule. However, according to other aspects of the invention the acylation may take place in another position in the insulin molecule, e.g. the α-amino group in position B1 or in position where the natural amino acid residue in the insulin molecule has been substituted with a lysine residue provided that B29 is changed from a lysine to another amino acid residue.

Thus in one aspect the acylated insulin is acylated either in the α-amino group in the B1 position or in a free ε-amino group of a lysine residue in the A- or B-chain of the insulin molecule.

In one aspect the insulin is acylated in the free ε-amino group of the lysine residue in position B29 of the insulin molecule.

The acyl group will be a liphophilic group and will typically be a fatty acid moiety having from about 6 to about 32 carbon atoms comprising at least one free carboxylic acid group or a group which is negatively charged at neutral pH. The fatty acid moiety will more typically have from 6 to 24, from 8 to 20, from 12 to 20, from 12-16, from 10-16, from 10-20, from 14-18 or from 14-16 carbon atoms.

In one aspect the pharmaceutical composition comprises at least one free carboxylic acid or a group which is negatively charged at neutral pH. In one aspect the pharmaceutical composition comprises an acyl group which is derived from a dicarboxylic fatty acid with from 4 to 32 carbon atoms.

In a further aspect the fatty acid moiety is derived from a dicarboxylic fatty acid with from about 6 to about 32, from 6 to 24, from 8 to 20, from 12 to 20, from 12-16, from 10-16, from 10-20, from 14-18 or from 14-16 carbon atoms.

In one aspect the pharmaceutical composition comprises an acyl group which is attached to the insulin via a linker group through amide bonds.

The acyl group may be attached directly to the free amino group in question. However, the acyl group may also be attached via amide bonds by a linker which links the free amino group in the insulin molecule and the acyl group in question together.

The acylated insulin will typically have at least one, or two additional negative net charge compared to human insulin and more typically it will have two additional negative charges. The additional negative charge may be provided by the free carboxylic acid group in the fatty acid or by the linker group which may comprise one or more amino acid residues of which at least one will contain a free carboxylic acid or a group which is negatively charged at neutral pH. In a further aspect the acyl group is derived from a dicarboxylic fatty acid.

In one aspect the pharmaceutical composition comprises an insulin wherein the insulin has a side chain attached either to the α-amino group of the N-terminal amino acid residue of the B chain or to an ε-amino group of a Lys residue present in the B chain of the parent insulin moiety via an amide bond, which side chain comprises at least one free carboxylic acid group or a group which is negatively charged at neutral pH, a fatty acid moiety with about 4 to about 32 carbon atoms in the carbon chain; and possible one or more linkers linking the individual components in the side chain together via amide bonds.

In one aspect the insulin molecule has a side chain attached to the ε-amino group of a Lys residue present in the B chain of the parent insulin, the side chain being of the general formula:

—W—X—Y-Z2

wherein W is:

an α-amino acid residue having a carboxylic acid group in the side chain which residue forms, with one of its carboxylic acid groups, an amide group together with ε-amino group of a Lys residue present in the B chain of the parent insulin;

a chain composed of two, three or four α-amino acid residues linked together via amide carbonyl bonds, which chain—via an amide bond—is linked to an ε-amino group of a Lys residue present in the B chain of the parent insulin, the amino acid residues of W being selected from the group of amino acid residues having a neutral side chain and amino acid residues having a carboxylic acid group in the side chain so that W has at least one amino acid residue which has a carboxylic acid group in the side chain; or

a covalent bond from X to an ε-amino group of a Lys residue present in the B chain of the parent insulin;

X is:

—CO—;

—CH(COOH)CO—;

—CO—N(CH2COOH)CH2CO—;

—CO—N(CH2COOH)CH2CON(CH2COOH)CH2CO—;

—CO —N(CH2CH2COOH)CH2CH2CO—;

—CO—N(CH2CH2COOH)CH2CH2CON(CH2CH2COOH)CH2CH2CO—;

—CO —NHCH(COOH)(CH2)4NHCO—;

—CO—N(CH2CH2COOH)CH2CO—; or

—CO—N(CH2COOH)CH2CH2CO—. that a) when W is an amino acid residue or a chain of amino acid residues, via a bond from the underscored carbon forms an amide bond with an amino group in W, or b) when W is a covalent bond, via a bond from the underscored carbonyl carbon forms an amide bond with an ε-amino group of a Lys residue present in the B chain of the parent insulin;

Y is:

—(CH2)m— where m is an integer in the range of 6 to 32;

a divalent hydrocarbon chain comprising 1, 2 or 3 —CH═CH— groups and a number of —CH2— groups sufficient to give a total number of carbon atoms in the chain in the range of 10 to 32; and

Z2 is:

—COOH;

—CO-Asp;

—CO-Glu;

—CO-Gly;

—CO-Sar;

—CH(COOH)2;

—N(CH2COOH)2;

—SO3H; or

—PO3H and any Zn2+ complexes thereof, provided that when W is a covalent bond and X is —CO—, then Z is different from —COOH.

In one aspect the B30 amino acid residue has been deleted and the acylated insulin is a desB30 insulin.

In one aspect W is an α-amino acid residue having from 4 to 10 carbon atoms and in a further aspect W is selected from the group consisting of α-Asp, β-Asp, α-Glu, γ-Glu, α-hGlu and δ-hGlu.

In one aspect X is —CO—.

In one aspect Z2 is —COOH.

The substructure Y of the side chain —W—X—Y— Z2 can be a group of the formula —(CH2)m— where m is an integer in the range of from 6 to 32, from 8 to 20, from 12 to 20, or from 12-16.

In one aspect, Y is a divalent hydrocarbon chain comprising 1, 2 or 3 —CH═CH— groups and a number of —CH2— groups sufficient to give a total number of carbon atoms in the chain in the range of from 6 to 32, from 10 to 32, from 12 to 20, or from 12-16.

In one aspect, Y is a divalent hydrocarbon chain of the formula —(CH2)vC6H4(CH2)w— wherein v and w are integers or one of them is zero so that the sum of v and w is in the range of from 6 to 30, from 10 to 20, or from 12-16.

In a further aspect W is selected from the group consisting of α-Asp, β-Asp, α-Glu, and γ-Glu; X is —CO— or —CH(COOH)CO; Y is —(CH2)m— where m is an integer in the range of 12-18 and Z2 is —COOH or —CH(COOH)2.

In one aspect the side chain may comprise at least one aromatic group or at least one difunctional PEG group. Hereinafter, the abbreviation “PEG” is used for polyethyleneglycol.

In one aspect of the invention the acylated insulin used in the pharmaceutical composition is having a formula

wherein Ins is the parent insulin moiety which via the α-amino group of the N-terminal amino acid residue of the B chain or an ε-amino group of a Lys residue present in the B chain of the insulin moiety is bound to the CO— group in the side chain via an amide bond;

X4 is

—(CH2), where n is 1, 2, 3, 4, 5 or 6;

NR, where R is hydrogen or —(CH2)p—COOH; —(CH2)p—SO3H; —(CH2)p—PO3H2; —(CH2)p—O—SO3H2; —(CH2)p—O—PO3H2; arylene substituted with 1 or 2 —(CH2)p—O—COOH groups; —(CH2)p-tetrazolyl, where p is an integer in the range of 1 to 6;

—(CR1R2)q—NR—CO—, where R1 and R2 independently of each other and independently for each value of q can be H, —COOH, or OH, q is 1-6 and R is defined as above;

—((CR3R4)q1—NR—CO)2-4—, where R3 and R4 independently of each other and independently for each value of q1 can be H, —COOH, or OH, q1 is 1-6 and R is defined as above; or

a bond W1 is arylene or heteroarylene, which may be substituted with one or two groups selected from the group consisting of —COOH, —SO3H, and —PO3H2 and tetrazolyl, or W1 is a bond; m is 0, 1, 2, 3, 4, 5 or 6;

X5 is

where R is defined as above; or

a bond;

Y1 is

—(CR1R2)q—NR—CO—, where R1 and R2 independently of each other and independently for each value of q can be H, —COOH, a bond or OH, q is 1-6; and R is defined as above;

NR where R is defined as above;

—((CR3R4)q1—NR—CO)2-4—, where R3 and R4 independently of each other and independently for each value of q1 can be H, —COOH, or OH, q1 is 1-6 and R is defined as above; or

a bond;

Q7 is

—(CH2)r where r is an integer from 4 to 22;

a divalent hydrocarbon chain comprising 1, 2 or 3 —CH═CH— groups and a number of —CH2— groups sufficient to give a total number of carbon atoms in the chain in the range of 4 to 22; or

wherein Q8-Q13 independently of each other can be O; S or a bond; where s, w, t and z independently of each other are zero or an integer from 1 to 10 so that the sum of s, w, t and z is in the range from 4 to 22, and v1, v2, and v3 independently of each other can be zero or 1, provided that when W1 is a bond then Q7 is not a divalent hydrocarbon chain of the formula —(CH2)v4C6H4(CH2)W1— wherein v4 and w1 are integers or one of them is zero so that the sum of v4 and w1 is in the range of 6 to 22; and

Z1 is:

—COOH;

—CO-Asp;

—CO-Glu;

—CO-Gly;

—CO-Sar;

—CH(COOH)2;

—N(CH2COOH)2;

—SO3H

—PO3H2;

—O—SO3H;

—O—PO3H2;

-tetrazolyl or

—O—W2,

where W2 is arylene or heteroarylene substituted with one or two groups selected from —COOH, —SO3H, and —PO3H2 and tetrazolyl;

provided that if W1 is a bond and v1, v2 and v3 are all zero and Q1-6 are all a bond, then Z1 is O—W2 and any Zn2+ complex thereof.

In one aspect of the invention W1 is phenylene. In one aspect W1 is 5-7 membered heterocyclic ring system comprising nitrogen, oxygen or sulphur. In one aspect W1 is a 5 membered heterocyclic ring system comprising at least one oxygen.

In one aspect of the invention Q7 is —(CH2)r— where r is an integer in the range of from 4 to 22, from 8- to 20, from 12 to 20 or from 14-18. In one aspect Q8, Q9, Q12 and Q13 are all bonds, v2 is 1 and v1 and v3 are zero. In one aspect Q10 and Q11 are oxygen.

In one aspect of the invention X4 and Y1 are a bonds and X5 is

where R is —(CH2)p—COOH, where p is 1-4.

In one aspect Z1 is —COOH.

In one aspect of the invention the acylated insulin of the pharmaceutical composition is selected from the group consisting of

In one aspect of the invention the acylated insulin present in the pharmaceutical composition is having a formula

wherein Ins is the parent insulin moiety which via the α-amino group of the N-terminal amino acid residue of the B chain or an ε-amino group of a Lys residue present in the B chain of the insulin moiety is bound to the CO— group in the side chain via an amide bond; each n is independently 0, 1, 2, 3, 4, 5 or 6; Q1, Q2, Q3, and Q4 independently of each other can be

(CH2CH2O)s—; (CH2CH2CH2O)—; (CH2CH2CH2CH2O)s—; (CH2CH2OCH2CH2CH2CH2O)s— or (CH2CH2CH2OCH2CH2CH2CH2O)s— where s is 1-20

—(CH2)r— where r is an integer from 4 to 22; or a divalent hydrocarbon chain comprising 1, 2 or 3 —CH═CH— groups and a number of —CH2— groups sufficient to give a total number of carbon atoms in the chain in the range of 4 to 22;

—(CH2)t— or —(CH2OCH2)t—, where t is an integer from 1 to 6;

—(CR1R2)q—, where R1 and R2 independently of each other can be H, —COOH, (CH2)16COOH and R1 and R2 can be different at each carbon, and q is 1-6,

—((CR3R4)q1)1—(NHCO—(CR3R4)q—NHCO)12—((CR3R4)q1)1 or —((CR3R4)q1)1—(CONH—(CR3R4)q1—CONH)1-2—((CR3R4)q1—)—, —((CR3R4)q1)1—(NHCO—(CR3R4)q1—CONH)1-2—((CR3R4)q1)1 or —((CR3R4)q1)1—(CONH—(CR3R4)q1—NHCO)1-2—((CR3R4)q1)1 where R3 and R4 independently of each other can be H, —COOH, and R3 and R4 can be different at each carbon, and q1 is 1-6, or

a bond;

with the proviso that Q1-Q4 are different;

X1, X2 and X3 are independently

O;

a bond; or

where R is hydrogen or —(CH2)p—COOH, —(CH2)p—SO3H, —(CH2)p—PO3H2, —(CH2)p—O—SO3H; —(CH2)p—O—PO3H2; or —(CH2)p-tetrazol-5-yl, where each p independently of the other p's is an integer in the range of 1 to 6; and

Z is:

—COOH;

—CO-Asp;

—CO-Glu;

—CO-Gly;

—CO-Sar;

—C H(COOH)2;

—N(CH2COOH)2;

—SO3H

—OSO3H

—OPO3H2

—PO3H2 or

-tetrazol-5-yl

and any Zn2+ complex thereof.

In one aspect of the invention s is in the range of 2-12, 2-4 or 2-3. In one aspect s is preferably 1.

The parent insulin molecule is human insulin or an analogue thereof. Non limiting analogues of human insulin is desB30 analogue; insulin analogues where the amino acid residue in position B30 is Lys and the amino acid residue in position B29 is any codable amino acid except Cys, Met, Arg and Lys; insulin analogues where the amino acid residue at position A21 is Asn and insulin analogues where the amino acid residue at position B3 is Lys and the amino acid residue at position B29 is Glu.

In another group of parent insulin analogues, the amino acid residue at position B28 is Asp. A specific example from this group of parent insulin analogues is AspB28 human insulin disclosed in EP 214826.

In another group of parent insulin analogues, the amino acid residue at position B28 is Lys and the amino acid residue at position B29 is Pro. A specific example from this group of parent insulin analogues is LysB28ProB29 human insulin.

In another group of parent insulin analogues the amino acid residue in position B30 is Lys and the amino acid residue in position B29 is any codable amino acid except Cys, Met, Arg and Lys. An example is an insulin analogue where the amino acid residue at position B29 is Thr and the amino acid residue at position B30 is Lys. A specific example from this group of parent insulin analogues is ThrB29LysB30 human insulin.

In another group of parent insulin analogues, the amino acid residue at position B3 is Lys and the amino acid residue at position B29 is Glu. A specific example from this group of parent insulin analogues is LysB3GluB29 human insulin.

The pharmaceutical composition according to the present invention will comprise a therapeutically effective amount of the acylated insulin together with a pharmaceutically acceptable carrier for the treatment of type 1 diabetes, type 2 diabetes and other states that cause hyperglycaemia in patients in need of such a treatment.

In a further aspect of the invention, there is provided a pharmaceutical composition for treating type 1 diabetes, type 2 diabetes and other states that cause hyperglycaemia in a patient in need of such a treatment, comprising a therapeutically effective amount of an acylated insulin derivative as defined above in mixture with an insulin or an insulin analogue which has a rapid onset of action, together with pharmaceutically acceptable carriers and additives.

Thus the pharmaceutical composition may comprise a mixture of two insulin components: one with a protracted insulin action, a basal insulin, and the other with a rapid onset of action, a bolus insulin. An example of such mixture is Insulin aspart, AspB28 human insulin in mixture with NεB29—(Nα-(HOOC(CH2)14CO)-γ-Glu) desB30 human insulin corresponding to LysB29Nε-hexadecandioyl-γ-Glu desB30 human insulin disclosed in WO 2005/012347. Another example of such a mixture is Lispro, LysB28ProB29 human insulin, in mixture with LysB29Nε-hexadecandioyl-γ-Glu desB30 human insulin. A third example of such a mixture is Glulisine, LysB3GluB29-human insulin, in mixture with LysB29Nε-hexadecandioyl-γ-Glu desB30 human insulin.

In one aspect of the invention at least 85% of the rapid acting insulin is present as rapid acting insulin hexamer or complexes with a smaller molecular weight than rapid acting insulin hexamers.

In one aspect of the invention at least 90, 92, 95, 96, 97, 98, 99, 99.5% of the rapid acting insulin is present as rapid acting insulin hexamer or complexes with a smaller molecular weight than rapid acting insulin hexamers.

The acylated insulin derivative and the rapid acting insulin analogue can be mixed in a molar ratio about 90%/10%; about 75%/25%, about 70%/30% about 50%/50%, about 25%/75%, about 30%/70% or about 10%/90%.

In one aspect the pharmaceutical composition according to the invention will have a pH between about 6.5 to about 8.5. In another aspect the pH is from about 7.0 to about 8.2, the pH is from about 7.2 to 8.0 or from about 7.4 to about 8.0 or the pH is from about 7.4 to about 7.8.

The invention further comprises a method for producing a pharmaceutical composition comprising an acylated insulin wherein more than about 4 zinc atoms per 6 molecules of acylated insulin are added to the composition. In a further aspect of the invention more than about 4.3 zinc atoms per 6 molecules of acylated insulin are added to the composition or more than about 4.5 zinc atoms per 6 molecules of acylated insulin are added to the composition or than about 5 zinc atoms per 6 molecules of acylated insulin are added to the composition. In a further aspect more than about 5.5 zinc atoms or more than about 6.5 zinc atoms, or more than about 7.0 zinc atoms or more than about 7.5 zinc atoms per 6 molecules of acylated insulin are added to the composition. In one aspect of the invention the method comprises adding up to about 12 zinc atoms per 6 molecules of acylated insulin to the composition. In one aspect of the invention the method comprises adding between about 4.3 and about 12 zinc atoms per 6 molecules of acylated insulin to the composition

In a further aspect of the invention between about 4.5 and about 12 zinc atoms per 6 molecules of acylated insulin are added to the composition or about 5 and about 11.4 zinc atoms per 6 molecules of acylated insulin are added to the composition or between about 5.5 and about 10 zinc atoms per 6 molecules of acylated insulin are added to the composition.

In a further aspect between about 6 and about 10.5 zinc atoms per 6 molecules of acylated insulin or between about 6.5 and about 10 zinc atoms per 6 molecules of acylated insulin or between about 7 and about 9 zinc atoms per 6 molecules of acylated insulin are added to the composition.

In one aspect of the invention the method comprises adding zinc to the composition before the addition of a preservative. In a further aspect of the invention the number of zinc atoms added before addition of a preservative is more than 1 zinc atom per 6 molecules of acylated insulin, or the number of zinc atoms added before addition of a preservative is more than 2 zinc atom per 6 molecules of acylated insulin or the number of zinc atoms added before addition of a preservative is more than 3 zinc atom per 6 molecules of acylated insulin or the number of zinc atoms added before addition of a preservative is more than 4 zinc atom per 6 molecules of acylated insulin or the number of zinc atoms added before addition of a preservative is more than 5 zinc atom per 6 molecules of acylated insulin.

In a further aspect of the invention between about 4.5 and about 12 zinc atoms per 6 molecules of acylated insulin are added to the composition before the addition of a preservative or about 5 and about 11.4 zinc atoms per 6 molecules of acylated insulin are added to the composition before the addition of a preservative or even more preferred between about 5.5 and about 10 zinc atoms per 6 molecules of acylated insulin are added to the composition before the addition of a preservative. In a further aspect between about 6 and about 10.5 zinc atoms per 6 molecules of acylated insulin or between about 6.5 and about 10 zinc atoms per 6 molecules of acylated insulin or between about 7 and about 9 zinc atoms per 6 molecules of acylated insulin are added to the composition before the addition of a preservative. In one aspect of the invention the method comprises adding zinc to the composition after addition of a preservative. In one aspect of the invention at least 0.5 zinc atom per 6 molecules of acylated insulin is added to the composition after addition of a preservative or at least 1 zinc atom per 6 molecules of acylated insulin is added to the composition after addition of a preservative.

In a further aspect of the invention more than about 2 zinc atoms per 6 molecules of acylated insulin are added to the composition after the addition of a preservative or more than about 3 zinc atoms per 6 molecules of acylated insulin are added to the composition after the addition of a preservative or more than about 4 zinc atoms per 6 molecules of acylated insulin are added to the composition after the addition of a preservative.

In a further aspect of the invention between about 0.5 and about 12, between about 1 and about 11.4, between about 1.5 and about 11, between about 2 and about 10.5, between about 3 and about 10 or between about 4 and about 9 zinc atoms per 6 molecules of acylated insulin are added to the composition after the addition of a preservative.

In a further aspect of the invention between about 4.5 and about 12 zinc atoms per 6 molecules of acylated insulin are added to the composition after the addition of a preservative or about 5 and about 11.4 zinc atoms per 6 molecules of acylated insulin are added to the composition after the addition of a preservative or between about 5.5 and about 10 zinc atoms per 6 molecules of acylated insulin are added to the composition after the addition of a preservative.

In a further aspect between about 6 and about 10.5 zinc atoms per 6 molecules of acylated insulin or between about 6.5 and about 10 zinc atoms per 6 molecules of acylated insulin or between about 7 and about 9 zinc atoms per 6 molecules of acylated insulin are added to the composition after the addition of a preservative.

In one aspect of the invention the method comprises adding part of the zinc before addition of a preservative and adding part of the zinc after addition of a preservative.

In one aspect the method comprises adding at least 0.5 zinc atom per 6 molecules of acylated insulin before addition of a preservative and adding at least 1 zinc atom per 6 molecules of acylated insulin after addition of a preservative. In one aspect the method comprises at least 0.5 zinc atom per 6 molecules of acylated insulin before addition of a preservative and adding at least 2, 3, 4, 5, 6, 7, 8, 9, 10 or 11 zinc atoms per 6 molecules of acylated insulin after addition of a preservative.

In one aspect the method comprises adding at least 1 zinc atom per 6 molecules of acylated insulin before addition of a preservative and adding at least 1 zinc atom per 6 molecules of acylated insulin after addition of a preservative or adding at least 1 zinc atom per 6 molecules of acylated insulin before addition of a preservative and adding at least 2 or 3 zinc atoms per 6 molecules of acylated insulin after addition of a preservative or adding at least 1 zinc atom per 6 molecules of acylated insulin before addition of a preservative and up to about 11 zinc atom per 6 molecules of acylated insulin after addition of a preservative. In one aspect the method comprises adding at least 1 zinc atom per 6 molecules of acylated insulin before addition of a preservative and adding at least 4, 5, 6, 7, 8, 9, 10 or 11 zinc atoms per 6 molecules of acylated insulin after addition of a preservative.

In one aspect the method comprises adding at least 2 zinc atoms per 6 molecules of acylated insulin before addition of a preservative and adding at least 1 zinc atom per 6 molecules of acylated insulin after addition of a preservative or adding at least 2 zinc atoms per 6 molecules of acylated insulin before addition of a preservative and adding at least 2 or 3 zinc atoms per 6 molecules of acylated insulin after addition of a preservative or adding at least 2 zinc atoms per 6 molecules of acylated insulin before addition of a preservative and up to about 10 zinc atoms per 6 molecules of acylated insulin after addition of a preservative In one aspect the method comprises adding at least 2 zinc atom per 6 molecules of acylated insulin before addition of a preservative and adding at least 4, 5, 6, 7, 8, 9 or 10 zinc atoms per 6 molecules of acylated insulin after addition of a preservative.

In one aspect the method comprises adding at least 3 zinc atoms per 6 molecules of acylated insulin before addition of a preservative and adding at least 1 zinc atom per 6 molecules of acylated insulin after addition of a preservative or adding at least 3 zinc atoms per 6 molecules of acylated insulin before addition of a preservative and adding at least 2 or 3 zinc atoms per 6 molecules of acylated insulin after addition of a preservative or adding at least 3 zinc atoms per 6 molecules of acylated insulin before addition of a preservative and up to about 9 zinc atoms per 6 molecules of acylated insulin after addition of a preservative

In one aspect of the invention the number of zinc atoms added before addition of a preservative is at least 3 zinc atom per 6 molecules of acylated insulin and the number of zinc atoms added after addition of a preservative are at least 3 zinc atoms per 6 molecules of acylated insulin. In one aspect the method comprises adding at least 3 zinc atom per 6 molecules of acylated insulin before addition of a preservative and adding at least 4, 5, 6, 7, 8 or 9 zinc atoms per 6 molecules of acylated insulin after addition of a preservative.

In one aspect of the invention the preservative added is phenol and/or m-cresol.

In one aspect of the invention the method comprises adding the whole amount of zinc atoms to the pharmaceutical composition in one step.

In one aspect of the invention the method comprises adding the zinc atoms to the pharmaceutical composition in two or more steps. For example, zinc may be added to the composition in one, two, three, four or five steps, where each step includes addition of small amounts of max. 1 Zn/6ins. The zinc may be added to the composition in one, two, three, four or five steps, where each step includes addition of small amounts of 2 Zn/6ins, 3 Zn/6ins, 4 Zn/6ins, 5 Zn/6ins or 6 Zn/6ins.

In one aspect of the invention, the method comprises adding a surfactant to the pharmaceutical composition. The surfactant can be mixed in the pharmaceutical composition in an amount of 0.0005-0.01% based on the weight of the pharmaceutical composition. In one aspect the surfactant can be mixed in the pharmaceutical composition in an amount of 0.0005-0.007% based on the weight of the composition. An example of a surfactant could be polysorbate 20, which can be mixed in the pharmaceutical composition in an amount of 0.001-0.003% based on the weight of the composition. Another example is poloxamer 188, which can be mixed in the pharmaceutical composition in an amount of 0.002-0.006% based on the weight of the composition.

In a further aspect of the invention the method comprises an acylated insulin which insulin has a side chain attached to the ε-amino group of a Lys residue present in the B chain of the parent insulin, the side chain being of the general formula:

—W—X—Y-Z2

wherein W is:

an α-amino acid residue having a carboxylic acid group in the side chain which residue forms, with one of its carboxylic acid groups, an amide group together with ε-amino group of a Lys residue present in the B chain of the parent insulin;

a chain composed of two, three or four α-amino acid residues linked together via amide carbonyl bonds, which chain—via an amide bond—is linked to an ε-amino group of a Lys residue present in the B chain of the parent insulin, the amino acid residues of W being selected from the group of amino acid residues having a neutral side chain and amino acid residues having a carboxylic acid group in the side chain so that W has at least one amino acid residue which has a carboxylic acid group in the side chain; or

a covalent bond from X to an ε-amino group of a Lys residue present in the B chain of the parent insulin;

X is:

—CO—;

—CH(COOH)CO—;

—CO—N(CH2COOH)CH2CO—;

—CO—N(CH2COOH)CH2CON(CH2COOH)CH2CO—;

—CO—N(CH2CH2COOH)CH2CH2CO—;

—CO—N(CH2CH2COOH)CH2CH2CON(CH2CH2COOH)CH2CH2CO—;

—CO—NHCH(COOH)(CH2)4NHCO—;

—CO—N(CH2CH2COOH)CH2CO—; or

—CO—N(CH2COOH)CH2CH2CO—. that a) when W is an amino acid residue or a chain of amino acid residues, via a bond from the underscored carbon forms an amide bond with an amino group in W, or b) when W is a covalent bond, via a bond from the underscored carbonyl carbon forms an amide bond with an ε-amino group of a Lys residue present in the B chain of the parent insulin;

Y is:

—(CH2)m— where m is an integer in the range of 6 to 32;

a divalent hydrocarbon chain comprising 1, 2 or 3 —CH═CH— groups and a number of —CH2— groups sufficient to give a total number of carbon atoms in the chain in the range of 10 to 32; and

wherein Ins is the parent insulin moiety which via the α-amino group of the N-terminal amino acid residue of the B chain or an ε-amino group of a Lys residue present in the B chain of the insulin moiety is bound to the CO— group in the side chain via an amide bond;

X4 is

—(CH2), where n is 1, 2, 3, 4, 5 or 6;

NR, where R is hydrogen or —(CH2)p—COOH; —(CH2)p—SO3H; —(CH2)p—PO3H2; —(CH2)p—O—SO3H2; —(CH2)p—O—PO3H2; arylene substituted with 1 or 2 —(CH2)p—O—COOH groups; —(CH2)p-tetrazolyl, where p is an integer in the range of 1 to 6;

—(CR1R2)q—NR—CO—, where R1 and R2 independently of each other and independently for each value of q can be H, —COOH, or OH, q is 1-6 and R is defined as above;

—((CR3R4)q1—NR—CO)2-4—, where R3 and R4 independently of each other and independently for each value of q1 can be H, —COOH, or OH, q1 is 1-6 and R is defined as above; or

a bond W1 is arylene or heteroarylene, which may be substituted with one or two groups selected from the group consisting of —COOH, —SO3H, and —PO3H2 and tetrazolyl, or W1 is a bond; m is 0, 1, 2, 3, 4, 5 or 6;

X5 is

where R is defined as above; or

a bond;

Y1 is

—(CR1R2)q—NR—CO—, where R1 and R2 independently of each other and independently for each value of q can be H, —COOH, a bond or OH, q is 1-6; and R is defined as above;

NR where R is defined as above;

—((CR3R4)q1—NR—CO)2-4—, where R3 and R4 independently of each other and independently for each value of q1 can be H, —COOH, or OH, q1 is 1-6 and R is defined as above; or

a bond;

Q7 is

—(CH2)r where r is an integer from 4 to 22;

a divalent hydrocarbon chain comprising 1, 2 or 3 —CH═CH— groups and a number of —CH2— groups sufficient to give a total number of carbon atoms in the chain in the range of 4 to 22; or

wherein Q8-Q13 independently of each other can be O; S or a bond; where s, w, t and z independently of each other are zero or an integer from 1 to 10 so that the sum of s, w, t and z is in the range from 4 to 22, and v1, v2, and v3 independently of each other can be zero or 1, provided that when W1 is a bond then Q7 is not a divalent hydrocarbon chain of the formula —(CH2)v4C6H4(CH2)w1— wherein v4 and w1 are integers or one of them is zero so that the sum of v4 and w1 is in the range of 6 to 22; and

Z1 is:

—COOH;

—CO-Asp;

—CO-Glu;

—CO-Gly;

—CO-Sar;

—CH(COOH)2;

—N(CH2COOH)2;

—SO3H

—PO3H2;

—O—SO3H;

—O—PO3H2;

-tetrazolyl or

—O—W2,

where W2 is arylene or heteroarylene substituted with one or two groups selected from —COOH, —SO3H, and —PO3H2 and tetrazolyl;

provided that if W1 is a bond and v1, v2 and v3 are all zero and Q8-13 are all a bonds, then Z1 is O—W2 and any Zn2+ complex thereof.

In one aspect of the invention W1 is phenylene. In one aspect W1 is 5-7 membered heterocyclic ring system comprising nitrogen, oxygen or sulphur. In one aspect W1 is a 5 membered heterocyclic ring system comprising at least one oxygen.

In one aspect of the invention Q7 is —(CH2)r— where r is an integer in the range of from 4 to 22, from 8- to 20, from 12 to 20 or from 14-18. In one aspect Q8, Q9, Q12 and Q13 are all bonds, v2 is 1 and v1 and v3 are zero. In one aspect Q10 and Q11 are oxygen.

In one aspect of the invention X4 and Y1 are a bonds and X5 is

where R is —(CH2)p—COOH, where p is 1-4.

In one aspect Z1 is —COOH.

In one aspect of the invention the acylated insulin of the method is selected from the group consisting of

In one aspect of the invention the acylated insulin used in the method is having a formula

wherein Ins is the parent insulin moiety which via the α-amino group of the N-terminal amino acid residue of the B chain or an ε-amino group of a Lys residue present in the B chain of the insulin moiety is bound to the CO— group in the side chain via an amide bond; each n is independently 0, 1, 2, 3, 4, 5 or 6; Q1, Q2, Q3, and Q4 independently of each other can be

(CH2CH2O)s—; (CH2CH2CH2O)—; (CH2CH2CH2CH2O)s—; (CH2CH2OCH2CH2CH2CH2O)s— or (CH2CH2CH2OCH2CH2CH2CH2O)s— where s is 1-20

—(CH2)r— where r is an integer from 4 to 22; or a divalent hydrocarbon chain comprising 1, 2 or 3 —CH═CH— groups and a number of —CH2— groups sufficient to give a total number of carbon atoms in the chain in the range of 4 to 22;

—(CH2)t— or —(CH2OCH2)t—, where t is an integer from 1 to 6;

—(CR1R2)q—, where R1 and R2 independently of each other can be H, —COOH, (CH2)16COOH and R1 and R2 can be different at each carbon, and q is 1-6,

—((CR3R4)q1)1—(NHCO—(CR3R4)q—NHCO)1-2—((CR3R4)q1)1 or —((CR3R4)q1)1—(CONH—(CR3R4)q1—CONH)1-2—((CR3R4)q1—)—, —((CR3R4)q1)1—(NHCO—(CR3R4)q1—CONH)1-2—((CR3R4)q1)1 or —((CR3R4)q1)1—(CONH—(CR3R4)q1—NHCO)1-2—((CR3R4)q1)1 where R3 and R4 independently of each other can be H, —COOH, and R3 and R4 can be different at each carbon, and q1 is 1-6, or

a bond;

with the proviso that Q1-Q4 are different;

X1, X2 and X3 are independently

O;

a bond; or

where R is hydrogen or —(CH2)p—COOH, —(CH2)p—SO3H, —(CH2)p—PO3H2, —(CH2)p—O—SO3H; —(CH2)p—O—PO3H2; or —(CH2)p-tetrazol-5-yl, where each p independently of the other p's is an integer in the range of 1 to 6; and

Z is:

—COOH;

—CO-Asp;

—CO-Glu;

—CO-Gly;

—CO-Sar;

—C H(COOH)2;

—N(CH2COOH)2;

—SO3H

—OSO3H

—OPO3H2

—PO3H2 or

-tetrazol-5-yl

and any Zn2+ complex thereof.

In one aspect of the invention s is in the range of 2-12, 2-4 or 2-3. In one aspect s is preferably 1.

In one aspect of the invention the method comprises adding a rapid acting insulin to the composition. The rapid acting insulin is AspB28human insulin, LysB28ProB29 human insulin and LysB3GluB29-human insulin or a mixture thereof.

In one aspect of the invention the pharmaceutical composition comprising acylated insulin is used for treatment of diabetes.

In one aspect of the invention, the pharmaceutical composition comprising acylated insulin is used for the manufacture of a medicament for the treatment of diabetes.

In one aspect the invention is related to a pharmaceutical composition according to the invention together with a pharmaceutically acceptable carrier and/or a pharmaceutically acceptable additive, which composition can be provided for the treatment of type 1 diabetes, type 2 diabetes and other states that cause hyperglycaemia in patients in need of such a treatment.

In one aspect of the invention, there is provided a method of treating type 1 diabetes, type 2 diabetes and other states that cause hyperglycaemia in a patient in need of such a treatment, comprising administering to the patient a therapeutically effective amount of an pharmaceutical composition together with a pharmaceutically acceptable carrier and/or pharmaceutical acceptable additives.

In one aspect of the invention, there is provided a method for the manufacture of a pharmaceutical composition for the use in the treatment of type 1 diabetes, type 2 diabetes and other states that cause hyperglycaemia.

In one aspect of the invention, there is provided a pharmaceutical composition for treating type 1 diabetes, type 2 diabetes and other states that cause hyperglycaemia in a patient in need of such a treatment.

In one aspect of the invention, there is provided a method of treating type 1 diabetes, type 2 diabetes and other states that cause hyperglycaemia in a patient in need of such a treatment, comprising administering to the patient a therapeutically effective amount of an pharmaceutical composition according to the invention.

In one aspect of the invention, there is provided a method for the manufacture of a pharmaceutical composition for the use in the treatment of type 1 diabetes, type 2 diabetes and other states that cause hyperglycaemia.

The pharmaceutical composition comprising an acylated insulin as defined in the pre-sent specification may be administered simultaneously or sequentially with OAD(s) or GLP-1. The factors may be supplied in single-dosage form wherein the single-dosage form contains both compounds, or in the form of a kit-of-parts comprising a preparation of a the pharmaceutical composition comprising a pharmaceutical composition comprising an acylated insulin and a pharmaceutical composition containing an OAD as a second unit dosage form. Whenever a first or second or third, etc., unit dose is mentioned throughout this specification this does not indicate the preferred order of administration, but is merely done for convenience purposes.

By “simultaneous” dosing of a preparation of a pharmaceutical composition comprising an acylated insulin and a preparation of OAD(s) or GLP-1 is meant administration of the compounds in single-dosage form, or administration of a first agent followed by administration of a second agent with a time separation of no more than 15 minutes, 10, 5 or 2 minutes. Either factor may be administered first.

By “sequential” dosing is meant administration of a first agent followed by administration of a second agent with a time separation of more than 15 minutes. Either of the two unit dosage form may be administered first. Preferably, both products are injected through the same intravenous access.

In a further aspect of the invention the pharmaceutical composition comprising an acylated insulin is administered once daily simultaneously or sequentially with OAD(s) or GLP-1. In a more preferred aspect the pharmaceutical composition comprising an acylated insulin further comprises a rapid acting insulin and is administered once daily together with OAD(s) or GLP-1. In one aspect of the invention the pharmaceutical composition comprising an acylated insulin can be a stand alone intensive treatment given up till 5 times daily. In an even more preferred aspect the pharmaceutical composition further comprises a rapid acting insulin, where the intensive treatment can be a stand alone treatment given up till 5 times daily.

The invention will be summarized in the following paragraphs:

1. A soluble pharmaceutical composition comprising an acylated insulin and further comprising more than 4 zinc atoms per 6 molecules of acylated insulin.

2. Pharmaceutical composition according to paragraph 1 comprising up to about 12 zinc atoms per 6 molecules of acylated insulin.

3. Pharmaceutical composition according to paragraphs 1 or 2 comprising between about 4.3 and about 12 zinc atoms per 6 molecules of acylated insulin.

4. Pharmaceutical composition according to any of paragraphs 1-3 comprising between about 4.5 and about 12 zinc atoms per 6 molecules of acylated insulin.

5. Pharmaceutical composition according to any of the preceding paragraphs, wherein at least 85% of the acylated insulin is present as complexes which are acylated insulin dodecamers or complexes with a higher molecular weight than acylated insulin dodecamer.

6. Pharmaceutical composition according to any of the preceding paragraphs, wherein at least 92% of the acylated insulin is present as complexes which are acylated insulin dodecamers or complexes with a higher molecular weight than acylated insulin dodecamer.

7. Pharmaceutical composition according to any of the preceding paragraphs, wherein at least 95% of the acylated insulin is present as complexes which are acylated insulin dodecamers or complexes with a higher molecular weight than acylated insulin dodecamer.

8. Pharmaceutical composition according to any of the preceding paragraphs, wherein at least 97% of the acylated insulin is present as complexes which are acylated insulin dodecamers or complexes with a higher molecular weight than acylated insulin dodecamer.

9. Pharmaceutical composition according to any of the preceding paragraphs, wherein the composition comprises a surfactant.

10. Pharmaceutical composition according to any of the preceding paragraphs, wherein the acylated insulin is an insulin acylated in the ε-amino group of a Lys residue in a position in the B-chain of the parent insulin molecule.

11. Pharmaceutical composition according to any of paragraphs 1-10, wherein the acyl group comprises at least one free carboxylic acid or a group which is negatively charged at neutral pH.

12. Pharmaceutical composition according to paragraph 1 or 10-11, wherein the acyl group is derived from a dicarboxylic fatty acid with from 4 to 32 carbon atoms.

13. Pharmaceutical composition according to paragraph 1 or 10-12, wherein the acyl group is attached to the insulin molecule via a linker group through amide bonds.

14. Pharmaceutical composition according to paragraph 1 or 13, wherein the linker group comprises at least one free carboxylic group or a group which is negatively charged at neutral pH.

15. Pharmaceutical composition according to any of paragraphs 1-14, wherein the insulin molecule has a side chain attached either to the α-amino group of the N-terminal amino acid residue of the B chain or to an ε-amino group of a Lys residue present in the B chain of the parent insulin moiety via an amide bond, which side chain comprises at least one free carboxylic acid group or a group which is negatively charged at neutral pH, a fatty acid moiety with about 4 to about 32 carbon atoms in the carbon chain; and possible one or more linkers linking the individual components in the side chain together via amide bonds.

16. Pharmaceutical composition according to paragraphs 1-15, wherein the side chain comprises at least one aromatic group.

17. Pharmaceutical composition according to paragraphs 1-15, wherein the side chain comprises at least one difunctional PEG group.

18. Pharmaceutical composition according to any of paragraphs 1-15, wherein the insulin molecule has a side chain attached to the ε-amino group of a Lys residue present in the B chain of the parent insulin, the side chain being of the general formula:

—W—X—Y-Z2

wherein W is:

an α-amino acid residue having a carboxylic acid group in the side chain which residue forms, with one of its carboxylic acid groups, an amide group together with ε-amino group of a Lys residue present in the B chain of the parent insulin;

a chain composed of two, three or four α-amino acid residues linked together via amide carbonyl bonds, which chain—via an amide bond—is linked to an ε-amino group of a Lys residue present in the B chain of the parent insulin, the amino acid residues of W being selected from the group of amino acid residues having a neutral side chain and amino acid residues having a carboxylic acid group in the side chain so that W has at least one amino acid residue which has a carboxylic acid group in the side chain; or

a covalent bond from X to an ε-amino group of a Lys residue present in the B chain of the parent insulin;

X is:

—CO—;

—CH(COOH)CO—;

—CO—N(CH2COOH)CH2CO—;

—CO—N(CH2COOH)CH2CON(CH2COOH)CH2CO—;

—CO—N(CH2CH2COOH)CH2CH2CO—;

—CO—N(CH2CH2COOH)CH2CH2CON(CH2CH2COOH)CH2CH2CO—;

—CO —NHCH(COOH)(CH2)4NHCO—;

—CO—N(CH2CH2COOH)CH2CO—; or

—CO—N(CH2COOH)CH2CH2CO—. that a) when W is an amino acid residue or a chain of amino acid residues, via a bond from the underscored carbon forms an amide bond with an amino group in W, or b) when W is a covalent bond, via a bond from the underscored carbonyl carbon forms an amide bond with an ε-amino group of a Lys residue present in the B chain of the parent insulin;

Y is:

—(CH2)m— where m is an integer in the range of 6 to 32;

a divalent hydrocarbon chain comprising 1, 2 or 3 —CH═CH— groups and a number of —CH2— groups sufficient to give a total number of carbon atoms in the chain in the range of 10 to 32; and

Z2 is:

—COOH;

—CO-Asp;

—CO-Glu;

—CO-Gly;

—CO-Sar;

—CH(COOH)2;

—N(CH2COOH)2;

—SO3H; or

—PO3H and any Zn2+ complexes thereof, provided that when W is a covalent bond and X is —CO—, then Z is different from —COOH.

19. Pharmaceutical composition according to any of paragraphs 1-15 and 18, wherein Z2 is —COOH.

21. Pharmaceutical composition according to paragraph 1-16, wherein the acylated insulin is having a formula

wherein Ins is the parent insulin moiety which via the α-amino group of the N-terminal amino acid residue of the B chain or an ε-amino group of a Lys residue present in the B chain of the insulin moiety is bound to the CO— group in the side chain via an amide bond;

X4 is

—(CH2), where n is 1, 2, 3, 4, 5 or 6;

NR, where R is hydrogen or —(CH2)p—COOH; —(CH2)p—SO3H; —(CH2)p—PO3H2; —(CH2)p—O—SO3H2; —(CH2)p—O—PO3H2; arylene substituted with 1 or 2 —(CH2)p—O—COOH groups; —(CH2)p-tetrazolyl, where p is an integer in the range of 1 to 6;

—(CR1R2)q—NR—CO—, where R1 and R2 independently of each other and independently for each value of q can be H, —COOH, or OH, q is 1-6 and R is defined as above;

—((CR3R4)q1—NR—CO)2-4—, where R3 and R4 independently of each other and independently for each value of q1 can be H, —COOH, or OH, q1 is 1-6 and R is defined as above; or

a bond W1 is arylene or heteroarylene, which may be substituted with one or two groups selected from the group consisting of —COOH, —SO3H, and —PO3H2 and tetrazolyl, or W1 is a bond; m is 0, 1, 2, 3, 4, 5 or 6;

X5 is

where R is defined as above; or

a bond;

Y1 is

—(CR1R2)q—NR—CO—, where R1 and R2 independently of each other and independently for each value of q can be H, —COOH, a bond or OH, q is 1-6; and R is defined as above;

NR where R is defined as above;

—((CR3R4)q1—NR—CO)2-4—, where R3 and R4 independently of each other and independently for each value of q1 can be H, —COOH, or OH, q1 is 1-6 and R is defined as above; or

a bond;

Q7 is

—(CH2)r where r is an integer from 4 to 22;

a divalent hydrocarbon chain comprising 1, 2 or 3 —CH═CH— groups and a number of —CH2— groups sufficient to give a total number of carbon atoms in the chain in the range of 4 to 22; or

wherein Q8-Q13 independently of each other can be O; S or a bond; where s, w, t and z independently of each other are zero or an integer from 1 to 10 so that the sum of s, w, t and z is in the range from 4 to 22, and v1, v2, and v3 independently of each other can be zero or 1, provided that when W1 is a bond then Q7 is not a divalent hydrocarbon chain of the formula —(CH2)v4C6H4(CH2)w1— wherein v4 and w1 are integers or one of them is zero so that the sum of v4 and w1 is in the range of 6 to 22; and

Z1 is:

—COOH;

—CO-Asp;

—CO-Glu;

—CO-Gly;

—CO-Sar;

—CH(COOH)2;

—N(CH2COOH)2;

—SO3H

—PO3H2;

—O—SO3H;

—O—PO3H2;

-tetrazolyl or

—O—W2,

where W2 is arylene or heteroarylene substituted with one or two groups selected from —COOH, —SO3H, and —PO3H2 and tetrazolyl;

provided that if W1 is a bond and v1, v2 and v3 are all zero and Q8-13 are all a bonds, then Z1 is O—W2 and any Zn2+ complex thereof.

22. Pharmaceutical composition according to paragraphs or 21, wherein W1 is phenylene.

30. Pharmaceutical composition to paragraph 1-15 and 17, wherein the acylated insulin is having a formula

wherein Ins is the parent insulin moiety which via the α-amino group of the N-terminal amino acid residue of the B chain or an ε-amino group of a Lys residue present in the B chain of the insulin moiety is bound to the CO— group in the side chain via an amide bond; each n is independently 0, 1, 2, 3, 4, 5 or 6; Q1, Q2, Q3, and Q4 independently of each other can be

(CH2CH2O)s—; (CH2CH2CH2O)s—; (CH2CH2CH2CH2O)s—; (CH2CH2OCH2CH2CH2CH2O)s— or (CH2CH2CH2OCH2CH2CH2CH2O)s— where s is 1-20

—(CH2)r— where r is an integer from 4 to 22; or a divalent hydrocarbon chain comprising 1, 2 or 3 —CH═CH— groups and a number of —CH2— groups sufficient to give a total number of carbon atoms in the chain in the range of 4 to 22;

—(CH2)t— or —(CH2OCH2)t—, where t is an integer from 1 to 6;

—(CR1R2)q—, where R1 and R2 independently of each other can be H, —COOH, (CH2)16COOH and R1 and R2 can be different at each carbon, and q is 1-6,

—((CR3R4)q1)1—(NHCO—(CR3R4)q—NHCO)1-2—((CR3R4)q1)1 or —((CR3R4)q1)1—(CONH—(CR3R4)q1—CONH)1-2—((CR3R4)q1—)—, —((CR3R4)q1)1—(NHCO—(CR3R4)q1—CONH)1-2—((CR3R4)q1)1 or —((CR3R4)q1)1—(CONH—(CR3R4)q1—NHCO)1-2—((CR3R4)q1)1 where R3 and R4 independently of each other can be H, —COOH, and R3 and R4 can be different at each carbon, and q1 is 1-6-, or

a bond;

with the proviso that Q1-Q4 are different;

X1, X2 and X3 are independently

O;

a bond; or

where R is hydrogen or —(CH2)p—COOH, —(CH2)p—SO3H, —(CH2)p—PO3H2, —(CH2)p—O—SO3H; —(CH2)p—O—PO3H2; or —(CH2)p-tetrazol-5-yl, where each p independently of the other p's is an integer in the range of 1 to 6; and

Z is:

—COOH;

—CO-Asp;

—CO-Glu;

—CO-Gly;

—CO-Sar;

—CH(COOH)2;

—N(CH2COOH)2;

—SO3H

—OSO3H

—OPO3H2

—PO3H2 or

-tetrazol-5-yl

and any Zn2+ complex thereof.

31. Pharmaceutical composition according to paragraphs 1 or 30, wherein s is in the range of 2-12, 2-4 or 2-3

38. Pharmaceutical composition according to any of the preceeding paragraphs, having a pH between about 6.5 and 8.5

39. Pharmaceutical composition according to any of the preceding paragraphs further comprising a rapid acting insulin

40. Pharmaceutical composition according to paragraphs 1 and 39, wherein at least 85% of the rapid acting insulin is present as rapid acting insulin hexamer or complexes with a smaller molecular weight than rapid acting insulin hexamers.

41. Pharmaceutical composition according to paragraphs 1 and 39-40, wherein at least 92% of the rapid acting insulin is present as rapid acting insulin hexamer or complexes with a smaller molecular weight than rapid acting insulin hexamers.

42. Pharmaceutical composition according to paragraphs 1 and 39-41, wherein at least 95% of the rapid acting insulin is present as rapid acting insulin hexamer or complexes with a smaller molecular weight than rapid acting insulin hexamers.

43. Pharmaceutical composition according to paragraphs 1 and 39-42, wherein at least 97% of the rapid acting insulin is present as rapid acting insulin hexamer or complexes with a smaller molecular weight than rapid acting insulin hexamers.

44. Pharmaceutical composition according to paragraphs 1 and 39-43, wherein at least 99% of the rapid acting insulin is present as rapid acting insulin hexamer or complexes with a smaller molecular weight than rapid acting insulin hexamers.

46. Method for producing a pharmaceutical composition comprising an acylated insulin wherein more than about 4 zinc atoms per 6 molecules of acylated insulin are added to the composition.

47. Method according to paragraph 46 wherein up to about 12 zinc atoms per 6 molecules of acylated insulin are added to the composition.

48. Method according to any of paragraphs 46-47 wherein between about 4.3 and about 12 zinc atoms per 6 molecules of acylated insulin are added to the composition.

49. Method according to paragraph 46-48 wherein the zinc is added to the composition before addition of a preservative.

50. Method according to any of the paragraphs 46-49 wherein the number of zinc atoms added before addition of a preservative is more than 1 zinc atom per 6 molecules of acylated insulin.

51. Method according to any of the paragraphs 46-50 wherein the number of zinc atoms added before addition of a preservative is more than 2 zinc atom per 6 molecules of acylated insulin.

52. Method according to any of the paragraphs 46-51 wherein the number of zinc atoms added before addition of a preservative is more than 3 zinc atom per 6 molecules of acylated insulin.

53. Method according to any of the paragraphs 46-52 wherein the number of zinc atoms added before addition of a preservative is more than 4 zinc atom per 6 molecules of acylated insulin.

54. Method according to any of the paragraphs 46-53 wherein the number of zinc atoms added before addition of a preservative is more than 5 zinc atom per 6 molecules of acylated insulin.

55. Method according to any of the paragraphs 46-48 wherein the zinc is added to the composition after addition of a preservative.

56. Method according to paragraph 46-48 and 55 wherein at least 0.5 zinc atom per 6 molecules of acylated insulin is added to the composition after addition of a preservative.

57. Method according to any of the paragraphs 46-48 and 55-56 wherein at least 1 zinc atom per 6 molecules of acylated insulin is added to the composition after addition of a preservative.

58. Method according to any of the preceding method paragraphs, wherein part of the zinc is added before addition of a preservative and part of the zinc is added after addition of a preservative

59. Method according to any of the preceding method paragraphs, wherein the number of zinc atoms added before addition of a preservative is at least 3 zinc atom per 6 molecules of acylated insulin and the number of zinc atoms added after addition of a preservative are at least 3 zinc atoms per 6 molecules of acylated insulin.

60. Method according to any of the preceding method paragraphs, wherein the preservative is phenol and/or m-cresol.

61. Method according to any of the preceding method paragraphs, wherein a surfactant is mixed with the pharmaceutical composition.

62. Method according to any of the preceding method paragraphs, wherein acylated insulin has a side chain attached to the ε-amino group of a Lys residue present in the B chain of the parent insulin, the side chain being of the general formula:

—W—X—Y-Z2

wherein W is:

an α-amino acid residue having a carboxylic acid group in the side chain which residue forms, with one of its carboxylic acid groups, an amide group together with ε-amino group of a Lys residue present in the B chain of the parent insulin;

a chain composed of two, three or four α-amino acid residues linked together via amide carbonyl bonds, which chain—via an amide bond—is linked to an ε-amino group of a Lys residue present in the B chain of the parent insulin, the amino acid residues of W being selected from the group of amino acid residues having a neutral side chain and amino acid residues having a carboxylic acid group in the side chain so that W has at least one amino acid residue which has a carboxylic acid group in the side chain; or

a covalent bond from X to an ε-amino group of a Lys residue present in the B chain of the parent insulin;

X is:

—CO—;

—CH(COOH)CO—;

—CO—N(CH2COOH)CH2CO—;

—CO—N(CH2COOH)CH2CON(CH2COOH)CH2CO—;

—CO—N(CH2CH2COOH)CH2CH2CO—;

—CO—N(CH2CH2COOH)CH2CH2CON(CH2CH2COOH)CH2CH2CO—;

—CO—NHCH(COOH)(CH2)4NHCO—;

—CO—N(CH2CH2COOH)CH2CO—; or

—CO—N(CH2COOH)CH2CH2CO—. that a) when W is an amino acid residue or a chain of amino acid residues, via a bond from the underscored carbon forms an amide bond with an amino group in W, or b) when W is a covalent bond, via a bond from the underscored carbonyl carbon forms an amide bond with an ε-amino group of a Lys residue present in the B chain of the parent insulin;

Y is:

—(CH2)m— where m is an integer in the range of 6 to 32;

a divalent hydrocarbon chain comprising 1, 2 or 3 —CH═CH— groups and a number of —CH2— groups sufficient to give a total number of carbon atoms in the chain in the range of 10 to 32; and

Z2 is:

—COOH;

—CO-Asp;

—CO-Glu;

—CO-Gly;

—CO-Sar;

—CH(COOH)2;

—N(CH2COOH)2;

—SO3H; or

—PO3H and any Zn2+ complexes thereof, provided that when W is a covalent bond and X is —CO—, then Z is different from —COOH.

64. Method according to paragraphs 46-61, wherein the acylated insulin is having a formula

wherein Ins is the parent insulin moiety which via the α-amino group of the N-terminal amino acid residue of the B chain or an ε-amino group of a Lys residue present in the B chain of the insulin moiety is bound to the CO— group in the side chain via an amide bond;

X4 is

—(CH2), where n is 1, 2, 3, 4, 5 or 6;

NR, where R is hydrogen or —(CH2)p—COOH; —(CH2)p—SO3H; —(CH2)p—PO3H2; —(CH2)p—O—SO3H2; —(CH2)p—O—PO3H2; arylene substituted with 1 or 2 —(CH2)p—O—COOH groups; —(CH2)p-tetrazolyl, where p is an integer in the range of 1 to 6;

—(CR1R2)q—NR—CO—, where R1 and R4 independently of each other and independently for each value of q can be H, —COOH, or OH, q is 1-6 and R is defined as above;

—((CR3R4)q1—NR—CO)2-4—, where R3 and R4 independently of each other and independently for each value of q1 can be H, —COOH, or OH, q1 is 1-6 and R is defined as above; or

a bond W1 is arylene or heteroarylene, which may be substituted with one or two groups selected from the group consisting of —COOH, —SO3H, and —PO3H2 and tetrazolyl, or W1 is a bond; m is 0, 1, 2, 3, 4, 5 or 6;

X5 is

where R is defined as above; or

a bond;

Y1 is

—(CR1R2)q—NR—CO—, where R1 and R2 independently of each other and independently for each value of q can be H, —COOH, a bond or OH, q is 1-6; and R is defined as above;

NR where R is defined as above;

—((CR3R4)q1—NR—CO)2-4—, where R3 and R4 independently of each other and independently for each value of q1 can be H, —COOH, or OH, q1 is 1-6 and R is defined as above; or

a bond;

Q7 is

—(CH2)r— where r is an integer from 4 to 22;

a divalent hydrocarbon chain comprising 1, 2 or 3 —CH═CH— groups and a number of —CH2— groups sufficient to give a total number of carbon atoms in the chain in the range of 4 to 22; or

wherein Q8-Q13 independently of each other can be O; S or a bond; where s, w, t and z independently of each other are zero or an integer from 1 to 10 so that the sum of s, w, t and z is in the range from 4 to 22, and v1, v2, and v3 independently of each other can be zero or 1, provided that when W1 is a bond then Q7 is not a divalent hydrocarbon chain of the formula —(CH2)v4C6H4(CH2)W1— wherein v4 and w1 are integers or one of them is zero so that the sum of v4 and w1 is in the range of 6 to 22; and

Z1 is:

—COOH;

—CO-Asp;

—CO-Glu;

—CO-Gly;

—CO-Sar;

—CH(COOH)2;

—N(CH2COOH)2;

—SO3H

—PO3H2;

O—SO3H;

O—PO3H2;

-tetrazolyl or

—O—W2,

where W2 is arylene or heteroarylene substituted with one or two groups selected from —COOH, —SO3H, and —PO3H2 and tetrazolyl;

provided that if W1 is a bond and v1, v2 and v3 are all zero and Q8-13 are all a bonds, then Z1 is O—W2 and any Zn2+ complex thereof.

65. Method according to paragraphs 46 and 64, wherein W1 is phenylene.

67. Method according to paragraph 66, wherein W1 is a 5 membered heterocyclic ring system comprising at least one oxygen.

68. Method according to paragraphs 46 and 64-67, wherein Q7 is —(CH2)r— where r is an integer in the range of from 4 to 22, from 8- to 20, from 12 to 20 or from 14-18.

69. Method according to paragraphs 46 and 64-68, wherein Q8, Q9, Q12 and Q13 are all a bonds, v2 is 1 and v1 and v3 are zero.

70. Method according paragraph 69, wherein Q10 and Q11 are oxygen.

71. Method according paragraph 64, wherein X4 and Y1 are a bond and X5 is

where R is —(CH2)p—COOH, where p is 1-4.

72. Method according to paragraph 64-71, wherein Z1 is —COOH.

73. Method according to paragraphs 46-61, wherein the acylated insulin is having a formula

wherein Ins is the parent insulin moiety which via the α-amino group of the N-terminal amino acid residue of the B chain or an ε-amino group of a Lys residue present in the B chain of the insulin moiety is bound to the CO— group in the side chain via an amide bond; each n is independently 0, 1, 2, 3, 4, 5 or 6; Q1, Q2, Q3, and Q4 independently of each other can be

(CH2CH2O)s—; (CH2CH2CH2O)s—; (CH2CH2CH2CH2O)s—; (CH2CH2OCH2CH2CH2CH2O)s— or (CH2CH2CH2OCH2CH2CH2CH2O)s— where s is 1-20

—(CH2)r— where r is an integer from 4 to 22; or a divalent hydrocarbon chain comprising 1, 2 or 3 —CH═CH— groups and a number of —CH2— groups sufficient to give a total number of carbon atoms in the chain in the range of 4 to 22;

—(CH2)t— or —(CH2OCH2)t—, where t is an integer from 1 to 6;

—(CR1R2)q—, where R1 and R2 independently of each other can be H, —COOH, (CH2)16COOH and R1 and R2 can be different at each carbon, and q is 1-6,

—((CR3R4)q1)1—(NHCO—(CR3R4)q—NHCO)1-2—((CR3R4)q1)1 or —((CR3R4)q1)1—(CONH—(CR3R4)q1—CONH)1-2—((CR3R4)q1—)—, —((CR3R4)q1)1—(NHCO—(CR3R4)q1—CONH)1-2—((CR3R4)q1)1 or —((CR3R4)q1)1—(CONH—(CR3R4)q1—NHCO)1-2—((CR3R4)q1)1 where R3 and R4 independently of each other can be H, —COOH, and R3 and R4 can be different at each carbon, and q1 is 1-6-, or

a bond;

with the proviso that Q1-Q4 are different;

X1, X2 and X3 are independently

O;

a bond; or

where R is hydrogen or —(CH2)p—COOH, —(CH2)p—SO3H, —(CH2)p—PO3H2, —(CH2)p—O—SO3H; —(CH2)p—O—PO3H2; or —(CH2)p-tetrazol-5-yl, where each p independently of the other p's is an integer in the range of 1 to 6; and

Z is:

—COOH;

—CO-Asp;

—CO-Glu;

—CO-Gly;

—CO-Sar;

—C H(COOH)2;

—N(CH2COOH)2;

—SO3H

—OSO3H

—OPO3H2

—PO3H2 or

-tetrazol-5-yl

and any Zn2+ complex thereof.

74. Method according to paragraph 73, wherein s is in the range of 2-12, 2-4 or 2-3

75. Method according to paragraph 73, wherein s is preferably 1.

76. Method according to paragraph 73-75, wherein Z is —COOH.

77. Method according to any of the preceding method paragraphs, wherein the parent insulin is a desB30 human insulin analogue.

83. Use of a composition according to any of the paragraphs 1-45 for the manufacture of a medicament for the treatment of diabetes.

84. A pharmaceutical composition for the treatment of diabetes in a patient in need of such treatment, comprising a therapeutically effective amount of a pharmaceutical composition according to paragraphs 1-45 together with a pharmaceutically acceptable carrier.

85. A method of treating diabetes in a patient in need of such a treatment, comprising administering to the patient a therapeutically effective amount of a pharmaceutical composition according to paragraphs 1-45 together with a pharmaceutically acceptable carrier.

86. A method according to paragraph 85 for pulmonary treatment of diabetes.

87. Use of a pharmaceutical composition according to paragraph 1-45 for the manufacture of a pharmaceutical composition for the use in the treatment of type 1 diabetes, type 2 diabetes and other states that cause hyperglycaemia.

88. Composition as described in the examples.

Another aspect of the invention is summarised in the following paragraphs.

91. A soluble pharmaceutical composition comprising an acylated insulin and further comprising more than 4 zinc atoms per 6 molecules of acylated insulin.

92. A soluble pharmaceutical composition according to paragraph 91 comprising an acylated insulin and further comprising more than 5 zinc atoms per 6 molecules of acylated insulin.

93. Pharmaceutical composition according to paragraphs 91-92 comprising up to about 14 zinc atoms per 6 molecules of acylated insulin.

94. Pharmaceutical composition according to paragraphs 91-93 comprising between about 5 and about 14 zinc atoms per 6 molecules of acylated insulin.

95. Pharmaceutical composition according to any of paragraphs 91-94 comprising between about 5 and about 13 zinc atoms per 6 molecules of acylated insulin.

96. Pharmaceutical composition according to any of paragraphs 91-95 comprising between about 5 and about 12 zinc atoms per 6 molecules of acylated insulin.

97. Pharmaceutical composition according to any of paragraphs 91-96 comprising between about 5.3 and about 12 zinc atoms per 6 molecules of acylated insulin.

98. Pharmaceutical composition according to any of paragraphs 91-97 comprising between about 5.5 and about 11.4 zinc atoms per 6 molecules of acylated insulin.

99. Pharmaceutical composition according to any of paragraphs 91-98 comprising between about 5.5 and about 10 zinc atoms per 6 molecules of acylated insulin.

100. Pharmaceutical composition according to any of paragraphs 91-99 wherein the acylated insulin is LysB29(Nε-tetradecanoyl)desB30 human insulin or LysB29Nε-lithocholoyl-γ-Glu desB30 human insulin

101. Pharmaceutical composition according to any of paragraphs 91-100 provided that the acylated insulin does not have a side chain attached to the ε-amino group of a Lys residue present in the B chain of the parent insulin, the side chain being of the general formula:

—W—X—Y-Z

wherein W is:

an α-amino acid residue having a carboxylic acid group in the side chain which residue forms, with one of its carboxylic acid groups, an amide group together with ε-amino group of a Lys residue present in the B chain of the parent insulin;

a chain composed of two, three or four α-amino acid residues linked together via amide carbonyl bonds, which chain—via an amide bond—is linked to an ε-amino group of a Lys residue present in the B chain of the parent insulin, the amino acid residues of W being selected from the group of amino acid residues having a neutral side chain and amino acid residues having a carboxylic acid group in the side chain so that W has at least one amino acid residue which has a carboxylic acid group in the side chain; or

a covalent bond from X to an ε-amino group of a Lys residue present in the B chain of the parent insulin;

X is:

—CO—;

—CH(COOH)CO—;

—CO—N(CH2COOH)CH2CO—;

—CO—N(CH2COOH)CH2CON(CH2COOH)CH2CO—;

—CO—N(CH2CH2COOH)CH2CH2CO—;

—CO—N(CH2CH2COOH)CH2CH2CON(CH2CH2COOH)CH2CH2CO—;

—CO—NHCH(COOH)(CH2)4NHCO—;

—CO—N(CH2CH2COOH)CH2CO—; or

—CO—N(CH2COOH)CH2CH2CO—. that a) when W is an amino acid residue or a chain of amino acid residues, via a bond from the underscored carbon forms an amide bond with an amino group in W, or b) when W is a covalent bond, via a bond from the underscored carbonyl carbon forms an amide bond with an ε-amino group of a Lys residue present in the B chain of the parent insulin;

Y is:

—(CH2)m— where m is an integer in the range of 6 to 32;

a divalent hydrocarbon chain comprising 1, 2 or 3 —CH═CH— groups and a number of —CH2— groups sufficient to give a total number of carbon atoms in the chain in the range of 10 to 32; and

Z is:

—COOH;

—CO-Asp;

—CO-Glu;

—CO-Gly;

—CO-Sar;

—CH(COOH)2;

—N(CH2COOH)2;

—SO3H; or

—PO3H.

101. Pharmaceutical composition according to any of paragraphs 91-101 provided that the acylated insulin is not LysB29Nε-hexadecandioyl-γ-Glu desB30 human insulin.

102. Pharmaceutical composition according to any of the preceeding paragraphs, having a pH between about 6.5 and 8.5

103. Pharmaceutical composition according to any of the preceding paragraphs further comprising a rapid acting insulin

109. Method for producing a pharmaceutical composition comprising an acylated insulin wherein more than about 4 zinc atoms per 6 molecules of acylated insulin are added to the composition.

In a further aspect of the invention more than about 4.3 zinc atoms per 6 molecules of acylated insulin are added to the composition or more than about 4.5 zinc atoms per 6 molecules of acylated insulin are added to the composition or than about 5 zinc atoms per 6 molecules of acylated insulin are added to the composition

110. Method according to paragraph 109 wherein up to about 14 zinc atoms per 6 molecules of acylated insulin are added to the composition.

111. Method according to any of paragraphs 109-110 wherein between about 4.3 and about 14 zinc atoms per 6 molecules of acylated insulin are added to the composition. In a further aspect of the invention between about 4.5 and about 12 zinc atoms per 6 molecules of acylated insulin are added to the composition or more preferred about 5 and about 11.4 zinc atoms per 6 molecules of acylated insulin are added to the composition or even more preferred between about 5.5 and about 10 zinc atoms per 6 molecules of acylated insulin are added to the composition

112. Method according to paragraph 109-111 wherein the zinc is added to the composition before addition of a preservative.

113. Method according to any of the paragraphs 109-112 wherein the number of zinc atoms added before addition of a preservative is more than 1 zinc atom per 6 molecules of acylated insulin.

114. Method according to any of the paragraphs 109-113 wherein the number of zinc atoms added before addition of a preservative is more than 2 zinc atoms per 6 molecules of acylated insulin.

115. Method according to any of the paragraphs 109-114 wherein the number of zinc atoms added before addition of a preservative is more than 3 zinc atoms per 6 molecules of acylated insulin.

116. Method according to any of the paragraphs 109-115 wherein the number of zinc atoms added before addition of a preservative is more than 4 zinc atoms per 6 molecules of acylated insulin.

117. Method according to any of the paragraphs 109-116 wherein the number of zinc atoms added before addition of a preservative is more than 5 zinc atoms per 6 molecules of acylated insulin.

In a further aspect of the invention between about 4.5 and about 12 zinc atoms per 6 molecules of acylated insulin are added to the composition before the addition of a preservative or more preferred about 5 and about 11.4 zinc atoms per 6 molecules of acylated insulin are added to the composition before the addition of a preservative or even more preferred between about 5.5 and about 10 zinc atoms per 6 molecules of acylated insulin are added to the composition before the addition of a preservative

118. Method according to any of the paragraphs 109-111 wherein the zinc is added to the composition after addition of a preservative.

119. Method according to paragraph 118 wherein at least 0.5 zinc atom per 6 molecules of acylated insulin is added to the composition after addition of a preservative.

120. Method according to any of the paragraphs 118-119 wherein at least 1 zinc atom per 6 molecules of acylated insulin is added to the composition after addition of a preservative.

In a further aspect of the invention more than about 2 zinc atoms per 6 molecules of acylated insulin are added to the composition after the addition of a preservative or more than about 3 zinc atoms per 6 molecules of acylated insulin are added to the composition after the addition of a preservative or more than about 4 zinc atoms per 6 molecules of acylated insulin are added to the composition after the addition of a preservative

In a further aspect of the invention between about 4.5 and about 12 zinc atoms per 6 molecules of acylated insulin are added to the composition after the addition of a preservative or more preferred about 5 and about 11.4 zinc atoms per 6 molecules of acylated insulin are added to the composition after the addition of a preservative or even more preferred between about 5.5 and about 10 zinc atoms per 6 molecules of acylated insulin are added to the composition after the addition of a preservative

121. Method according to any of the paragraphs 109-120 wherein part of the zinc is added before addition of a preservative and part of the zinc is added after addition of a preservative

In one aspect the method comprises adding at least 1 zinc atom per 6 molecules of acylated insulin before addition of a preservative and adding at least 1 zinc atom per 6 molecules of acylated insulin after addition of a preservative or adding at least 1 zinc atom per 6 molecules of acylated insulin before addition of a preservative and adding at least 2-3 zinc atoms per 6 molecules of acylated insulin after addition of a preservative or adding at least 1 zinc atom per 6 molecules of acylated insulin before addition of a preservative and up to about 11 zinc atom per 6 molecules of acylated insulin and after addition of a preservative

In one aspect the method comprises adding at least 2 zinc atoms per 6 molecules of acylated insulin before addition of a preservative and adding at least 1 zinc atom per 6 molecules of acylated insulin after addition of a preservative or adding at least 2 zinc atoms per 6 molecules of acylated insulin before addition of a preservative and adding at least 2-3 zinc atoms per 6 molecules of acylated insulin after addition of a preservative or adding at least 2 zinc atoms per 6 molecules of acylated insulin before addition of a preservative and up to about 10 zinc atoms per 6 molecules of acylated insulin after addition of a preservative

In one aspect the method comprises adding at least 3 zinc atoms per 6 molecules of acylated insulin before addition of a preservative and adding at least 1 zinc atom per 6 molecules of acylated insulin after addition of a preservative or adding at least 3 zinc atoms per 6 molecules of acylated insulin before addition of a preservative and adding at least 2-3 zinc atoms per 6 molecules of acylated insulin after addition of a preservative or adding at least 3 zinc atoms per 6 molecules of acylated insulin before addition of a preservative and up to about 9 zinc atoms per 6 molecules of acylated insulin after addition of a preservative

122. Method according to any of the paragraph 121 wherein the number of zinc atoms added before addition of a preservative is at least 3 zinc atom per 6 molecules of acylated insulin and the number of zinc atoms added after addition of a preservative are at least 3 zinc atoms per 6 molecules of acylated insulin.

123. Method according to any of the paragraphs 112-122 wherein the preservative is phenol and/or m-cresol.

124. Method according to any of the paragraphs 109-123 wherein the acylated insulin is LysB29(Nε-tetradecanoyl)desB30 human insulin or LysB29Nε-lithocholoyl-γ-Glu desB30 human insulin

125. Method according to any of the paragraphs 109-124 provided that the acylated insulin does not have a side chain attached to the ε-amino group of a Lys residue present in the B chain of the parent insulin, the side chain being of the general formula:

—W—X—Y-Z

wherein W is:

an α-amino acid residue having a carboxylic acid group in the side chain which residue forms, with one of its carboxylic acid groups, an amide group together with ε-amino group of a Lys residue present in the B chain of the parent insulin;

a chain composed of two, three or four α-amino acid residues linked together via amide carbonyl bonds, which chain—via an amide bond—is linked to an ε-amino group of a Lys residue present in the B chain of the parent insulin, the amino acid residues of W being selected from the group of amino acid residues having a neutral side chain and amino acid residues having a carboxylic acid group in the side chain so that W has at least one amino acid residue which has a carboxylic acid group in the side chain; or

a covalent bond from X to an ε-amino group of a Lys residue present in the B chain of the parent insulin;

X is:

—CO—;

—CH(COOH)CO—;

—N(CH2COOH)CH2CO—;

—N(CH2COOH)CH2CON(CH2COOH)CH2CO—;

—N(CH2CH2COOH)CH2CH2CO—;

—N(CH2CH2COOH)CH2CH2CON(CH2CH2COOH)CH2CH2CO—;

—NHCH(COOH)(CH2)4NHCO—;

—N(CH2CH2COOH)CH2CO—; or

—N(CH2COOH)CH2CH2CO—. that a) when W is an amino acid residue or a chain of amino acid residues, via a bond from the underscored carbon forms an amide bond with an amino group in W, or b) when W is a covalent bond, via a bond from the underscored carbonyl carbon forms an amide bond with an ε-amino group of a Lys residue present in the B chain of the parent insulin;

Y is:

—(CH2)m— where m is an integer in the range of 6 to 32;

a divalent hydrocarbon chain comprising 1, 2 or 3 —CH═CH— groups and a number of —CH2— groups sufficient to give a total number of carbon atoms in the chain in the range of 10 to 32; and

Z is:

—COOH;

—CO-Asp;

—CO-Glu;

—CO-Gly;

—CO-Sar;

—CH(COOH)2;

—N(CH2COOH)2;

—SO3H; or

—PO3H.

126. Method according to any of the paragraphs 125 provided that the acylated insulin is not LysB29Nε-hexadecandioyl-γ-Glu desB30 human insulin.

127. Method according to any of the paragraphs 109-126 wherein a rapid acting insulin is added to the composition.

129. Use of a composition according to any of the paragraphs 91-104 for the manufacture of a medicament for the treatment of diabetes.

BRIEF DESCRIPTION OF THE DRAWINGS

All figures are the result of size exclusion chromatography of a formulation of LysB29Nε-hexadecandioyl-γ-Glu desB30 human insulin and/or insulin aspart on Superose 6HR eluted by isotonic saline at 37° C. in the upper panels and the content of the respective 14 fractions covering from the high molecular weight fractions to low molecular weight fractions or insulin monomer fractions in the lower panel.

DETAILED DESCRIPTION OF THE INVENTION

The present invention is based on the surprising recognition that an increase in the content of zinc above the usual level (between 2 and 4 zinc atoms per six molecules of acylated insulin) increases the proportion of medium and high molecular weight insulin complexes of certain acylated insulin derivatives, in particular the acylated insulin LysB29Nε-hexadecandioyl-γ-Glu desB30 human insulin.

According to the present invention it is possible to design the insulin formulation containing the insulin with desired degree of association.

Furthermore, when mixing the acylated insulin with rapid acting insulin analogues a true biphasic action profile is obtained as no blunting occurs. Thus the invention provides soluble compositions that are mixtures of a rapid-acting insulin analogue and a prolonged-acting acylated insulin in which the rate of disappearance of the rapid acting insulin and the acylated insulin from the site of injection are the same as when injected in separate compositions. By administering insulin as a biphasic pharmaceutical composition, the number of injections can be reduced resulting in a more convenient and safe therapy.

The present invention is further based on the surprisingly recognition that, when preparing an insulin formulation, zinc can be added to the formulation after addition of preservatives. The zinc is typically provided by adding zinc acetate, zinc chloride or zinc citrate to the insulin formulation.

According to the invention, an acylated insulin composition of this invention may be delivered by inhalation to achieve rapid absorption thereof. Administration by inhalation can result in pharmacokinetics comparable to subcutaneous administration of insulins. Inhalation of an acylated insulin composition of this invention leads to a rapid rise in the level of circulating insulin followed by a rapid fall in blood glucose levels. Different inhalation devices typically provide similar pharmacokinetics when similar particle sizes and similar levels of lung deposition are compared.

As those skilled in the art will recognize, the formulation of an acylated insulin composition of this invention, the quantity of the formulation delivered, and the duration of administration of a single dose depend on the type of inhalation device employed. For some aerosol delivery systems, such as nebulizers, the frequency of administration and length of time for which the system is activated will depend mainly on the concentration of insulin conjugate in the aerosol. For example, shorter periods of administration can be used at higher concentrations of insulin conjugate in the nebulizer solution. Devices such as metered dose inhalers can produce higher aerosol concentrations, and can be operated for shorter periods to deliver the desired amount of insulin conjugate. Devices such as powder inhalers deliver active agent until a given charge of agent is expelled from the device. In this type of inhaler, the amount of insulin derivative of this invention in a given quantity of the powder determines the dose delivered in a single administration.

The particle size of insulin derivative of this invention in the formulation delivered by the inhalation device is critical with respect to the ability of insulin to make it into the lungs, and preferably into the lower airways or alveoli. Preferably, an acylated insulin composition of this invention is formulated so that at least about 10% of the insulin conjugate delivered is deposited in the lung, preferably about 10 to about 20%, or more. It is known that the maximum efficiency of pulmonary deposition for mouth breathing humans is obtained with particle sizes of about 2 μm to about 3 μm. When particle sizes are above about 5 μm pulmonary deposition decreases substantially. Particle sizes below about 1 μm cause pulmonary deposition to decrease, and it becomes difficult to deliver particles with sufficient mass to be therapeutically effective. Thus, particles of an acylated insulin composition delivered by inhalation have a particle size preferably less than about 10 μm, more preferably in the range of about 1 μm to about 5 μm. The formulation of the insulin derivative is selected to yield the desired particle size in the chosen inhalation device.

Advantageously for administration as a dry powder, an acylated insulin composition of this invention is prepared in a particulate form with a particle size of less than about 10 μm, preferably about 1 to about 5 μm. The preferred particle size is effective for delivery to the alveoli of the patient's lung. Preferably, the dry powder is largely composed of particles produced so that a majority of the particles have a size in the desired range. Advantageously, at least about 50% of the dry powder is made of particles having a diameter less than about 10 μm. Such formulations can be achieved by spray drying, milling, or critical point condensation of a solution containing insulin conjugate and other desired ingredients. Other methods also suitable for generating particles useful in the current invention are known in the art.

The particles are usually separated from a dry powder formulation in a container and then transported into the lung of a patient via a carrier air stream. Typically, in current dry powder inhalers, the force for breaking up the solid is provided solely by the patient's inhalation. In another type of inhaler, air flow generated by the patient's inhalation activates an impeller motor which deagglomerates the particles.

Formulations of acylated insulin of this invention for administration from a dry powder inhaler typically include a finely divided dry powder containing the acylated insulin, but the powder can also include a bulking agent, carrier, excipient, another additive, or the like. Additives can be included in a dry powder formulation of insulin conjugate, for example, to dilute the powder as required for delivery from the particular powder inhaler, to facilitate processing of the formulation, to provide advantageous powder properties to the formulation, to facilitate dispersion of the powder from the inhalation device, to stabilize the formulation (for example, antioxidants or buffers), to provide taste to the formulation, or the like. Advantageously, the additive does not adversely affect the patient's airways. The an acylated insulin can be mixed with an additive at a molecular level or the solid formulation can include particles of the insulin conjugate mixed with or coated on particles of the additive. Typical additives include mono-, di-, and polysaccharides; sugar alcohols and other polyols, such as, for example, lactose, glucose, raffinose, melezitose, lactitol, maltitol, trehalose, sucrose, mannitol, starch, or combinations thereof; surfactants, such as sorbitols, diphosphatidyl choline, or lecithin; or the like. Typically an additive, such as a bulking agent, is present in an amount effective for a purpose described above, often at about 50% to about 90% by weight of the formulation. Additional agents known in the art for formulation of a protein such as insulin analogue protein can also be included in the formulation.

A spray including the acylated insulin composition of this invention can be produced by forcing a suspension or solution of insulin conjugate through a nozzle under pressure. The nozzle size and configuration, the applied pressure, and the liquid feed rate can be chosen to achieve the desired output and particle size. An electrospray can be produced, for example, by an electric field in connection with a capillary or nozzle feed. Advantageously, particles of insulin conjugate delivered by a sprayer have a particle size less than about 10 μm, preferably in the range of about 1 μm to about 5 μm.

Formulations of acylated insulin of this invention suitable for use with a sprayer will typically include the insulin derivative in an aqueous solution at a concentration of about 1 mg to about 20 mg of insulin conjugate per ml of solution. The formulation can include agents such as an excipient, a buffer, an isotonicity agent, a preservative, a surfactant, and, preferably, zinc. The formulation can also include an excipient or agent for stabilization of the insulin derivative, such as a buffer, a reducing agent, a bulk protein, or a carbohydrate. Bulk proteins useful in formulating insulin conjugates include albumin, protamine, or the like. Typical carbohydrates useful in formulating insulin conjugates include sucrose, mannitol, lactose, trehalose, glucose, or the like. The insulin derivative formulation can also include a surfactant, which can reduce or prevent surface-induced aggregation of the insulin conjugate caused by atomization of the solution in forming an aerosol. Various conventional surfactants can be employed, such as polyoxyethylene fatty acid esters and alcohols, and polyoxyethylene sorbitol fatty acid esters. Amounts will generally range between about 0.001 and about 4% by weight of the formulation.

The pharmaceutical compositions according to the present invention may be administered parenterally to patients in need of such a treatment. Parenteral administration may be performed by subcutaneous, intramuscular or intravenous injection by means of a syringe, optionally a pen-like syringe or another convenient dosing equipment. Alternatively, parenteral administration can be performed by means of an infusion pump.

One aspect of the invention is related to a pharmaceutical composition according to the invention together with a pharmaceutically acceptable carrier and/or a pharmaceutically acceptable additive, which can be provided for pulmonary treatment of type 1 diabetes, type 2 diabetes and other states that cause hyperglycaemia in patients in need of such a treatment.

In one aspect of the invention, there is provided a method for the manufacture of a pharmaceutical composition for the use in the treatment of type 1 diabetes, type 2 diabetes and other states that cause hyperglycaemia, the composition being used pulmonary and comprising a therapeutically effective amount of a pharmaceutical composition according to the invention together with a pharmaceutically acceptable carrier and/or pharmaceutical acceptable additives.

Injectable compositions of the acylated insulin derivatives can be prepared using the conventional techniques of the pharmaceutical industry which involve dissolving and mixing the ingredients as appropriate to give the desired end product. Thus, according to one procedure, the insulin derivative is dissolved in an amount of water which is somewhat less than the final volume of the composition to be prepared. An isotonic agent, a preservative or a mixture of preservatives, zinc as acetate, citrate or chloride or a mixture thereof, and a buffer can be added as required, furthermore a surfactant might be added and the pH value of the solution is adjusted—if necessary—using an acid, e.g. hydrochloric acid, or a base, e.g. aqueous sodium hydroxide as needed. Finally, the volume of the solution is adjusted with water to give the desired concentration of the ingredients.

In a further aspect of the invention the formulation further comprises a pharmaceutically acceptable preservative which may be selected from the group consisting of phenol, o-cresol, m-cresol, p-cresol, methyl p-hydroxybenzoate, propyl p-hydroxybenzoate, 2-phenoxyethanol, butyl p-hydroxybenzoate, 2-phenylethanol, benzyl alcohol, chlorobutanol, and bronopol, benzoic acid, imidurea, chlorohexidine, sodium dehydroacetate, chlorocresol, ethyl p-hydroxybenzoate, benzethonium chloride, chlorphenesine (3-(4-chlorophenoxy)propane-1,2-diol) or mixtures thereof. In a further aspect of the invention the preservative is present in a concentration from 0.1 mg/ml to 20 mg/ml. In a further aspect of the invention the preservative is present in a concentration from 0.1 mg/ml to 5 mg/ml. In a further aspect of the invention the preservative is present in a concentration from 5 mg/ml to 10 mg/ml. In a further aspect of the invention the preservative is present in a concentration from 10 mg/ml to 20 mg/ml. Each one of these specific preservatives constitutes an alternative aspect of the invention. The use of a preservative in pharmaceutical compositions is well-known to the skilled person. For convenience reference is made to Remington: The Science and Practice of Pharmacy, 19th edition, 1995.

In a further aspect of the invention the formulation further comprises an isotonic agent which may be selected from the group consisting of a salt (e.g. sodium chloride), a sugar or sugar alcohol, an amino acid (e.g. 1-glycine, 1-histidine, arginine, lysine, isoleucine, aspartic acid, tryptophan, threonine), 1,2-propanediol (propyleneglycol), 1,3-propanediol, 1,3-butanediol) polyethyleneglycol (e.g. PEG400), or mixtures thereof. Any sugar such as mono-, di-, or polysaccharides, or water-soluble glucans, including for example fructose, glucose, mannose, sorbose, xylose, maltose, lactose, sucrose, trehalose, dextran, pullulan, dextrin, cyclodextrin, soluble starch, hydroxyethyl starch and carboxymethylcellulose-Na may be used. In one aspect the sugar additive is sucrose. Sugar alcohol is defined as a C4-C8 hydrocarbon having at least one —OH group and includes, for example, mannitol, sorbitol, inositol, galactitol, dulcitol, xylitol, and arabitol. In one aspect the sugar alcohol additive is mannitol. The sugars or sugar alcohols mentioned above may be used individually or in combination. There is no fixed limit to the amount used, as long as the sugar or sugar alcohol is soluble in the liquid composition and does not adversely effect the stabilizing effects achieved using the methods of the invention. In one aspect, the sugar or sugar alcohol concentration is between about 1 mg/ml and about 150 mg/ml. In a further aspect of the invention the isotonic agent is present in a concentration from 1 mg/ml to 50 mg/ml. In a further aspect of the invention the isotonic agent is present in a concentration from 1 mg/ml to 7 mg/ml. In a further aspect of the invention the isotonic agent is present in a concentration from 8 mg/ml to 24 mg/ml. In a further aspect of the invention the isotonic agent is present in a concentration from 25 mg/ml to 50 mg/ml. Each one of these specific isotonic agents constitutes an alternative aspect of the invention. The use of an isotonic agent in pharmaceutical compositions is well-known to the skilled person. For convenience reference is made to Remington: The Science and Practice of Pharmacy, 19th edition, 1995.

In a further aspect of the invention the formulation comprises a surfactant to prevent fibrillation especially when mixing the insulin derivative with a rapid acting insulin as insulin aspart. Various conventional surfactants can be employed, such as polyoxyethylene fatty acid esters and alcohols, and polyoxyethylene sorbitol fatty acid esters. Amounts will generally range between about 0.001 and about 0.1% by weight of the formulation.

The pharmaceutical compositions according to the present invention can be used in the treatment of states which are sensitive to insulin. Thus, they can be used in the treatment of type 1 diabetes, type 2 diabetes and hyperglycaemia for example as sometimes seen in seriously injured persons and persons who have undergone major surgery. The optimal dose level for any patient will depend on a variety of factors including the efficacy of the specific acylated insulin or mixture of the acylated insulin with a rapid acting insulin employed, the age, body weight, physical activity, and diet of the patient, on a possible combination with other drugs, and on the severity of the state to be treated. It is recommended that the daily dosage of the insulin derivative of this invention be determined for each individual patient by those skilled in the art in a similar way as for known insulin compositions.

The starting product for preparing the acylated insulin or insulin analogue contained in the composition according to the invention can be produced by either well-known peptide synthesis or by well known recombinant production in suitable transformed microorganisms. Thus the insulin starting product can be produced by a method which comprises culturing a host cell containing a DNA sequence encoding the polypeptide and capable of expressing the polypeptide in a suitable nutrient medium under conditions permitting the expression of the peptide, after which the resulting peptide is recovered from the culture.

The medium used to culture the cells may be any conventional medium suitable for growing the host cells, such as minimal or complex media containing appropriate supplements. Suitable media are available from commercial suppliers or may be prepared according to published recipes (e.g. in catalogues of the American Type Culture Collection). The peptide produced by the cells may then be recovered from the culture medium by conventional procedures including separating the host cells from the medium by centrifugation or filtration, precipitating the proteinaceous components of the supernatant or filtrate by means of a salt, e.g. ammonium sulphate, purification by a variety of chromatographic procedures, e.g. ion exchange chromatography, gel filtration chromatography, affinity chromatography, or the like, dependent on the type of peptide in question.

The DNA sequence encoding the insulin polypeptide may suitably be of genomic or cDNA origin, for instance obtained by preparing a genomic or cDNA library and screening for DNA sequences coding for all or part of the polypeptide by hybridisation using synthetic oligonucleotide probes in accordance with standard techniques (see, for example, Sambrook, J, Fritsch, E F and Maniatis, T, Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory Press, New York, 1989). The DNA sequence encoding the polypeptide may also be pre-pared synthetically by established standard methods, e.g. the phosphoamidite method described by Beaucage and Caruthers, Tetrahedron Letters 22 (1981), 1859-1869, or the method described by Matthes et al., EMBO Journal 3 (1984), 801-805. The DNA sequence may also be prepared by polymerase chain reaction using specific primers, for instance as described in U.S. Pat. No. 4,683,202 or Saiki et al., Science 239 (1988), 487-491.

The DNA sequence may be inserted into any vector which may conveniently be subjected to recombinant DNA procedures, and the choice of vector will often depend on the host cell into which it is to be introduced. Thus, the vector may be an autonomously replicating vector, i.e. a vector which exists as an extrachromosomal entity, the replication of which is independent of chromosomal replication, e.g. a plasmid. Alternatively, the vector may be one which, when introduced into a host cell, is integrated into the host cell genome and replicated together with the chromosome(s) into which it has been integrated.

The vector is preferably an expression vector in which the DNA sequence encoding the peptide is operably linked to additional segments required for transcription of the DNA, such as a promoter. The promoter may be any DNA sequence which shows transcriptional activity in the host cell of choice and may be derived from genes encoding proteins either homologous or heterologous to the host cell. Examples of suitable promoters for directing the transcription of the DNA encoding the peptide of the invention in a variety of host cells are well known in the art, cf. for instance Sambrook et al., Molecular Cloning—a laboratory manual, second edition 1989.

The DNA sequence encoding the peptide may also, if necessary, be operably connected to a suitable terminator, polyadenylation signals, transcriptional enhancer sequences, and translational enhancer sequences. The recombinant vector of the invention may further comprise a DNA sequence enabling the vector to replicate in the host cell in question.

The vector may also comprise a selectable marker, e.g. a gene the product of which complements a defect in the host cell or one which confers resistance to a drug, e.g. ampicillin, kanamycin, tetracyclin, chloramphenicol, neomycin, hygromycin or methotrexate.

To direct the insulin peptide into the secretory pathway of the host cells, a secretory signal sequence (also known as a leader sequence, prepro sequence or pre sequence) may be provided in the recombinant vector. The secretory signal sequence is joined to the DNA sequence encoding the peptide in the correct reading frame. Secretory signal sequences are commonly positioned 5′ to the DNA sequence encoding the peptide. The secretory signal sequence may be that normally associated with the peptide or may be from a gene encoding another secreted protein.

The procedures used to ligate the DNA sequences coding for the present peptide, the promoter and optionally the terminator and/or secretory signal sequence, respectively, and to insert them into suitable vectors containing the information necessary for replication, are well known to persons skilled in the art (cf., for instance, Sambrook et al., supra).

The host cell into which the DNA sequence or the recombinant vector is introduced may be any cell which is capable of producing the present peptide and includes bacteria, yeast, fungi and higher eukaryotic cells. Examples of suitable host cells well known and used in the art are, without limitation, E. coli, Saccharomyces cerevisiae, or mammalian BHK or CHO cell lines.

DEFINITIONS

The term “effective amount” as used herein means a dosage which is sufficient in order for the treatment of the patient to be effective compared with no treatment.

The term “pharmaceutical composition” as used herein means a product comprising an active compound or a salt thereof together with pharmaceutical excipients such as buffer, preservative and tonicity modifier, said pharmaceutical composition being useful for treating, preventing or reducing the severity of a disease or disorder by administration of said pharmaceutical composition to a person. Thus a pharmaceutical composition is also known in the art as a pharmaceutical formulation. It is to be understood that pH of a pharmaceutical composition which is to be reconstituted is the pH value which is measured on the reconstituted composition produced by reconstitution in the prescribed reconstitution liquid at room temperature.

The term “pharmaceutically acceptable” as used herein means suited for normal pharmaceutical applications, i.e. giving rise to no adverse events in patients etc.

The term “buffer” as used herein refers to a chemical compound in a pharmaceutical composition that reduces the tendency of pH of the composition to change over time as would otherwise occur due to chemical reactions. Buffers include chemicals such as sodium phosphate, TRIS, glycyl glycine and sodium citrate.

The term “preservative” as used herein refers to a chemical compound which is added to a pharmaceutical composition to prevent or delay microbial activity (growth and metabolism). Examples of pharmaceutically acceptable preservatives are phenol, m-cresol and a mixture of phenol and m-cresol.

The term “isotonicity agent” as used refers to a chemical compound in a pharmaceutical composition that serves to modify the osmotic pressure of the pharmaceutical composition so that the osmotic pressure becomes closer to that of human plasma. Isotonicity agents include NaCl, glycerol, mannitol etc.

The term “stabilizer” as used herein refers to chemicals added to peptide containing pharmaceutical compositions in order to stabilize the peptide, i.e. to increase the shelf life and/or in-use time of such compositions. Examples of stabilizers used in pharmaceutical formulations are L-glycine, L-histidine, arginine, polyethylene glycol, and carboxymethylcellulose. Further phenols, zinc ions and sodium chloride can act as stabilizers.

The term “surfactant” as used herein refers to a chemical compound in a pharmaceutical composition that serves to modify the interface to air and hydrophobic surfaces in a way that displaces or partly displaces insulin, insulin analogues and insulin derivatives from the interfaces. Various conventional surfactants can be employed, such as polyoxyethylene fatty acid esters and alcohols, and polyoxyethylene sorbitol fatty acid esters. An example is polysorbate 20.

The term “treatment of a disease” as used herein means the management and care of a patient having developed the disease, condition or disorder. The purpose of treatment is to combat the disease, condition or disorder. Treatment includes the administration of the active compounds to eliminate or control the disease, condition or disorder as well as to alleviate the symptoms or complications associated with the disease, condition or disorder.

The term “prevention of a disease” as used herein is defined as the management and care of an individual at risk of developing the disease prior to the clinical onset of the disease. The purpose of prevention is to combat the development of the disease, condition or disorder, and includes the administration of the active compounds to prevent or delay the onset of the symptoms or complications and to prevent or delay the development of related diseases, conditions or disorders.

The term “human insulin” as used herein means the human hormone whose structure and properties are well known. Human insulin has two polypeptide chains that are connected by disulphide bridges between cysteine residues, namely the A-chain and the B-chain. The A-chain is a 21 amino acid peptide and the B-chain is a 30 amino acid peptide, the two chains being connected by three disulphide bridges: one between the cysteines in position 6 and 11 of the A-chain, the second between the cysteine in position 7 of the A-chain and the cysteine in position 7 of the B-chain, and the third between the cysteine in position 20 of the A-chain and the cysteine in position 19 of the B-chain.

The term “basal insulin” as used herein means an formulation of insulin peptide which has a time-action of more than 15 hours in standard models of diabetes and is suited to cover the need for insulin during the night and in-between meals. Preferably, the basal insulin has a time-action of at least 20 hours. Preferably, the basal insulin has a time-action of at least 10 hours. Preferably, the basal insulin has a time-action in the range from 15 to 48 hours. Preferably, the basal insulin has a time-action similar to that observed for commercial pharmaceutical compositions of NPH insulin or NεB29-tetradecanoyl desB30 human insulin.

The term “bolus insulin”, “meal-related insulin” or “rapid acting insulin” as used herein means an insulin peptide which is rapid-acting and suited to cover the need for insulin during and after the meal.

The term “biphasic insulin” as used herein means a pharmaceutical composition comprising a mixture of “bolus insulin” and “basal insulin”.

With “desB30” or “B(1-29)” is meant an insulin B chain or an analogue thereof lacking the B30 amino acid residue and “A(1-21)” means the natural insulin A chain or an analogue thereof. The C-peptide and its amino acid sequence are indicated in the three letter amino acid code. DesB30, desB29 human insulin is a human insulin lacking B29 and B30.

With “B1”, “A1” etc. is meant the amino acid residue in position 1 in the B chain of insulin (counted from the N-terminal end) and the amino acid residue in position 1 in the A chain of insulin (counted from the N-terminal end), respectively. The amino acid residue in a specific position may also be denoted as e.g. PheB1 which means that the amino acid residue in position B1 is a phenylalanine residue.

By “insulin analogue” as used herein is meant a polypeptide which has a molecular structure which formally can be derived from the structure of a naturally occurring insulin, for example that of human insulin by deleting and/or exchanging at least one amino acid residue in the naturally occurring insulin and/or adding at least one amino acid residue.

The added and/or exchanged amino acid residues can either be codable amino acid residues or other naturally occurring residues or purely synthetic amino acid residues The insulin analogues may be such wherein position 28 of the B chain may be modified from the natural Pro residue to one of Asp, Lys, or Ile. In another aspect Lys at position B29 is modified to Pro. In one aspect B30 may be Lys and then B29 can be any codable amino acid except Cys, Met, Arg and Lys.

Also, Asn at position A21 may be modified to Ala, Gln, Glu, Gly, His, Ile, Leu, Met, Ser, Thr, Trp, Tyr or Val, in particular to Gly, Ala, Ser, or Thr and preferably to Gly. Furthermore, Asn at position B3 may be modified to Lys or Asp. Further examples of insulin analogues are desB30 human insulin; desB30 human insulin analogues; insulin analogues wherein PheB1 has been deleted; insulin analogues wherein the A-chain and/or the B-chain have an N-terminal extension and insulin analogues wherein the A-chain and/or the B-chain have a C-terminal extension. Thus one or two Arg may be added to position B1 or to position B30.

In one aspect an insulin analogue comprises less than 6 modifications (substitutions, deletions, additions) relative to the native peptide. In another aspect an analogue comprises less than 5 modifications (substitutions, deletions, additions) relative to the native peptide. In another aspect an analogue comprises less than 4 modifications (substitutions, deletions, additions) relative to the native peptide. In another aspect an analogue comprises less than 3 modifications (substitutions, deletions, additions) relative to the native peptide. In another aspect an analogue comprises less than 2 modifications (substitutions, deletions, additions) relative to the native peptide. In another aspect an analogue comprises only a single modification (substitutions, deletions, additions) relative to the native peptide.

By “insulin derivative” as used herein is meant a naturally occurring insulin or an insulin analogue which has been chemically modified, e.g. by introducing a side chain in one or more positions of the insulin backbone or by oxidizing or reducing groups of the amino acid residues in the insulin or by converting a free carboxylic group to an ester group or acylating a free amino group or a hydroxy group.

By “acylated insulin” as used herein is meant a naturally occurring insulin, for example that of human insulin, an insulin molecule, an insulin derivative or an insulin analogue which has been chemically modified, by acylating a free amino group or a hydroxy group.

The term “no blunting” as used herein means that when formulated in one formulation both the rapid acting insulin and the acylated insulin has profile of action which is identical or substantially identical with the profile of action, when administering the rapid acting insulin and the acylated insulin in separate formulations.

The term “OAD” or “OAD(s)” as used herein means oral antidiabetic drug or oral antidiabetic drugs. An unlimited list of OAD(s) can be sulfonylurea (SU), biguanides e.g. Melformin or thiozolidindiones (TZD).

The expression “a codable amino acid” or “a codable amino acid residue” is used to indicate an amino acid or amino acid residue which can be coded for by a triplet (“codon”) of nucleotides.

hGlu is homoglutamic acid.

α-Asp is the L-form of —HNCH(CO—)CH2COOH.

β-Asp is the L-form of —HNCH(COOH)CH2CO—.

α-Glu is the L-form of —HNCH(CO—)CH2CH2COOH.

γ-Glu is the L-form of —HNCH(COOH)CH2CH2CO—.

α-hGlu is the L-form of —HNCH(CO—)CH2CH2CH2COOH.

δ-hGlu is the L-form of —HNCH(COOH)CH2CH2CH2CO—.

β-Ala is —NH—CH2—CH2—COOH.

Sar is sarcosine (N-methylglycine).

The expression “an amino acid residue having a carboxylic acid group in the side chain” designates amino acid residues like Asp, Glu and hGlu. The amino acids can be in either the L- or D-configuration. If nothing is specified it is understood that the amino acid residue is in the L configuration.

When an insulin derivative according to the invention is stated to be “soluble at physiological pH values” it means that the insulin derivative can be used for preparing injectable insulin compositions that are fully dissolved at physiological pH values. Such favourable solubility may either be due to the inherent properties of the insulin derivative alone or a result of a favourable interaction between the insulin derivative and one or more ingredients contained in the vehicle.

The expression “high molecular weight insulin” or “hmw” means that the molecular weight of a complex of human insulin, of an insulin analogue or of an insulin derivative is above human serum albumin, above a dodecameric complex of an insulin analogue or of an insulin derivative or more than about 72 kDalton.

The expression “medium molecular weight insulin” or “mmw” means that the molecular weight of a complex of human insulin, of an insulin analogue or of an insulin derivative is from about an insulin hexamer to about an insulin dodecamer between 24 and 80 kDalton

The expression “low molecular weight insulin” or “lmw” means that the molecular weight of a human insulin, an insulin analogue or an insulin derivative is below 24 kDalton The expression “net charge” means the overall charge of the molecule. At pH 7.4, human insulin has a negative net charge about −3 or when forming a hexamer about −2.5 per insulin monomer.

The following abbreviations have been used in the specification and examples:

hGlu homoglutamic acid

Sar: Sarcosine (N-methyl-glycine)

S.c. subcutaneous

Acyl ins Acylated insulin

Ins insulin

All references, including publications, patent applications, and patents, cited herein are hereby incorporated by reference in their entirety and to the same extent as if each reference were individually and specifically indicated to be incorporated by reference and were set forth in its entirety herein (to the maximum extent permitted by law).

All headings and sub-headings are used herein for convenience only and should not be construed as limiting the invention in any way.

The use of any and all examples, or exemplary language (e.g., “such as”) provided herein, is intended merely to better illuminate the invention and does not pose a limitation on the scope of the invention unless otherwise claimed. No language in the specification should be construed as indicating any non-claimed element as essential to the practice of the invention.

The citation and incorporation of patent documents herein is done for convenience only and does not reflect any view of the validity, patentability, and/or enforceability of such patent documents.

This invention includes all modifications and equivalents of the subject matter recited in the claims appended hereto as permitted by applicable law.

Formulations of LysB29Nε-hexadecandioyl-γ-Glu desB30 human insulin 600 μM and Zn/6 acyl-ins varied at 3, 4, 6, and 8 were obtained by dilution of the above mentioned formulation B, D, F, and H with an equal amount of medium. The formulations were tested by size exclusion chromatography for the ability to form high molecular weight insulin on a Superose 6HR column eluted with 140 mM NaCl, 10 mM trishydroxymethylaminomethan pH 7.4, and 0.01% NaN3 at 37° C. and 0.25 mL/min. Fractions were collected for every 4 minutes starting at the high molecular weight exclusion limit and ending with the last of the monomer peak, in total 14 fractions. The fraction concentration of insulin analogue was quantified specifically by reverse phase chromatography and the concentration of zinc after addition of the chromophoric zinc chelator terpy at an pH 2.5 excursion (FIGS. 3,4,5 and 6). The relative amount of LysB29Nε-hexadecandioyl-γ-Glu desB30 human insulin with a size larger than albumin (fraction [1-8]) was measured at 3, 4, 6, and 8 Zn/6 insulin to 67.4, 88.9, 98.0, and 97.9% respectively. Furthermore the zinc concentration was following LysB29Nε-hexadecandioyl-γ-Glu desB30 human insulin especially in the high molecular size fractions.

Formulation consisting of insulin aspart 600 μM (A and E) varied at 3 and 6 zinc/insulin eluted as monomer (fraction[12-13]) only followed by minor part of the added zinc, see FIGS. 1-2.

Mixing of an Insulin Analogue and an Acylated Insulin

Formulations consisting of insulin 180 μM aspart and 420 μM LysB29Nε-hexadecandioyl-γ-Glu desB30 human insulin and zinc concentration varied at 3, 6, and 8 Zn/6insulin were obtained by mixing the above stock solution A to H and the mentioned medium in an analogous manner. An example of calculating the concentration of zinc/6 acylated insulin is mixing 180 μM aspart with 3 Zn/6insulin and 420 μM LysB29Nε-hexadecandioyl-γ-Glu desB30 human insulin with 3 Zn/6acyl-ins makes 4.3 Zn/6 acylated insulin [(180*3+420*3)/420=4.3 Zn/acyl-ins]. The formulations were tested by size exclusion chromatography for the ability to form high molecular weight insulin on a Superose 6HR column, and fractions were similarly collected for quantification of both insulin analogues and zinc. As seen from FIG. 8 the analogues mixing with 3 Zn/6insulin corresponding to a formulation of 4.3 Zn/6 acyl-insulin showed predominantly LysB29Nε-hexadecandioyl-γ-Glu desB30 human insulin (72.5% of LysB29Nε-hexadecandioyl-γ-Glu desB30 human insulin) and zinc in the high molecular weight fractions [1-8], but also insulin aspart, whereas an equal amount of insulin aspart and LysB29Nε-hexadecandioyl-γ-Glu desB30 human insulin was measured as mean in the rest of the fractions [9-14](FIG. 8). The testing of insulin aspart and LysB29Nε-hexadecandioyl-γ-Glu desB30 human insulin consisting of 6 Zn/6ins are shown in FIG. 2 and FIG. 5 respectively. Mixing the two formulations of 6 Zn/6ins (FIG. 2. and FIG. 5) to aspart 180 μM+LysB29Nε-hexadecandioyl-γ-Glu desB30 human insulin 420 μM corresponding to 8.6 Zn/6acyl-ins showed a completely separated fraction of LysB29Nε-hexadecandioyl-γ-Glu desB30 human insulin at the high molecular weight and insulin aspart at low molecular weight (FIG. 10). Mixing at equal concentration of insulin aspart and LysB29Nε-hexadecandioyl-γ-Glu desB30 human insulin to 12 Zn/6acyl-ins showed separated fractions as well (FIG. 12). Further increased concentration of zinc to 8 Zn/6ins in the individual formulations is shown in for LysB29Nε-hexadecandioyl-γ-Glu desB30 human insulin in FIG. 6 and the mix of aspart 180 μM+LysB29Nε-hexadecandioyl-γ-Glu desB30 human insulin 420 μM in FIG. 11.

As is clear from the figures, the increased amount of zinc surprisingly induced a separation of insulin aspart and LysB29Nε-hexadecandioyl-γ-Glu desB30 human insulin in the pharmaceutical preparation, resulting in no significant blunting between the analogues.

an increased amount of zinc induced a separation of insulin aspart and LysB29Nε-hexadecandioyl-γ-Glu desB30 human insulin in the pharmaceutical preparation in a way that the analogues showed actually no blunting.

8.2 mg LysB29Nε-hexadecandioyl-γ-Glu desB30 human insulin was dissolved in 400 μL water and 3 μCi 125I-TyrA14-LysB29Nε-hexadecandioyl-γ-Glu desB30 human insulin tracer was added, followed by 800 μL 4% glycerol and treated for 10-15 min in a vacuum centrifuge to remove added ethanol included in the tracer solution. 60 μL 10 mM Zn(AcO)2 was added and with 2 minutes interval followed by 100 μL 0.32 M phenol, 200 μL 0.16 M m-cresol, 80 μL 0.5 M NaCl, and 140 μL 0.1 M sodium phosphate and pH adjusted to pH 7.5 mM and a total volume of 2 mL. The solution was filtered through a sterile 0.22 μm filter and used for a disappearance study after subcutaneous injection in pig. T50%±SEM (h) was determined to 9.6±0.7 h as described under assay in a cross over study of formulation examples 2A to 2D.

8.2 mg LysB29Nε-hexadecandioyl-γ-Glu desB30 human insulin was dissolved in 400 μL water and 3 μCi 125I-TyrA14-LysB29Nε-hexadecandioyl-γ-Glu desB30 human insulin tracer was added, followed by 800 μL 4% glycerol and treated for 10-15 min in a vacumcentrifuge to remove added ethanol included in the tracer solution. 60 μL 10 mM Zn(AcO)2 was added and with 2 minutes interval followed by 100 μL 0.32 M phenol, 200 μL 0.16 M m-cresol, 60 μL 10 mM Zn(AcO)2, 80 μL 0.5 M NaCl, and 140 μL 0.1 M sodium phosphate and adjusted to pH 7.5 mM and a total volume of 2 mL. The solution was filtered through a sterile 0.22 μm filter and used for a disappearance study after subcutaneous injection in pig. T50%±SEM (h) was determined to 11.9±1.0 h as described under assay in a cross over study of formulation examples 2A to 2D.

2C: 3 Zn/6 Acylated Insulin Added Before and 3 Zn/6 Acylated Insulin After Phenol and m-cresol and Not Included Buffer

8.2 mg LysB29Nε-hexadecandioyl-γ-Glu desB30 human insulin was dissolved in 400 μL water and 3 μCi 125I-TyrA14-LysB29Nε-hexadecandioyl-γ-Glu desB30 human insulin tracer was added, followed by 800 μL 4% glycerol and treated for 10-15 min in a vacumcentrifuge to remove added ethanol included in the tracer solution. 60 μL 10 mM Zn(AcO)2 was added and with 2 minutes interval followed by 100 μL 0.32 M phenol, 200 μL 0.16 M m-cresol, 60 μL 10 mM Zn(AcO)2, 80 μL 0.5 M NaCl, and adjusted to pH 7.5 mM. The solution was filtered through a sterile 0.22 μm filter and used for a disappearance study after subcutaneous injection in pig. T50%±SEM (h) was determined to 14.4±2.4 h as described under assay in a cross over study of formulation examples 2A to 2D.

2D: 6 Zn/6 Acylated Insulin Added After Phenol and m-cresol and Not Included Buffer

8.2 mg LysB29Nε-hexadecandioyl-γ-Glu desB30 human insulin was dissolved in 400 μL water and 3 μCi 125I-TyrA14-LysB29Nε-hexadecandioyl-γ-Glu desB30 human insulin tracer was added, followed by 800 μL 4% glycerol and treated for 10-15 min in a vacuum centrifuge to remove added ethanol included in the tracer solution. 100 μL 0.32 M phenol was added and with 2 minutes interval followed by 200 μL 0.16 M m-cresol, 120 μL 10 mM Zn(AcO)2, 80 μL 0.5 M NaCl, and adjusted to pH 7.5 mM and a total volume of 2 mL. The solution was filtered through a sterile 0.22 μm filter and used for a disappearance study after subcutaneous injection in pig. T50%±SEM (h) was determined to 13.5±1.7 h as described under assay in a cross over study of formulation examples 2A to 2D.

Example 3

LysB29Nε-hexadecandioyl-γ-Glu desB30 human insulin can be crystallized as a hexamer with a zinc content of 2-6 mol zinc per 6 mol insulin and an excess of a phenol-like molecule (preferred phenol). As precipitants, ionic salts are used with NaCl being preferred. Furthermore crystallisation can be enhanced by adding small amounts of organic solvents (ethanol).

Preparation of a Protein Solution (Solution A):

To 1.8 ml of this LysB29Nε-hexadecandioyl-γ-Glu desB30 human insulin solution is added 69.5 μl of a solution containing 0.010 mol/l Zn(OAc)2 in water, and 18 μl of a solution containing 2 mol/l Phenol in ethanol.

Preparation of the Precipitant Solution (Solution B):

To a 5 ml of a solution containing 0.1M trishydroxymethylaminomethan (adjusted to pH 7.5 with HCl) is added 1.75 g NaCl and 1 ml Ethanol and the final volume adjusted to 10 ml.

Crystallisation:

Equal amounts (typically 500 μl) of solution A and solution B are mixed in a glass vial. Crystallisation is complete after 12 h at room temperature and the resulting crystals can be isolated by filtration or centrifugation.

KAV area peak1 is measured from KAV=0 to KAV=0.46 (32 min) as relative area % of total area for KAV<0.46 corresponding to self association larger than albumin. For KAV peak 1 about 0.56 (albumin size) integration cut is between albumin size and insulin hexamer size.

SEC Method for Mix of Long Acting Insulin Derivative and Rapid Acting Insulin Aspart Using Specific Detection of Peak Fractions:

Miscibility of Insulin Aspart (3Zn/6ins) and prolonged acting insulin derivative 50:50, as measured by collecting fractions from SEC and quantifying by HPLC the presence of fast-acting and prolonged-acting insulins in the high molecular weight fraction (peak1) and in the low molecular weight fraction (peak2). Fraction cut is following integration cut mentioned above.

Conclusion: SEC (Size Exclusion Chromatography) using isotonic saline eluent at body temperature is used as a model for insulin self association after injection in subcutis when phenolic preservatives have disappeared. Including phenol in the eluent SEC evaluates the state of insulin self association in the pharmaceutical formulation and briefly upon injection.

B29Nε-hexadecandioyl-γ-Glu desB30 insulin 600 μM was formulated with zinc concentrations increasing stepwise by one from 0 to 6 Zn/6ins, and the SEC method showed self association to very high molecular weight and more than human albumin for more than 90%, when the zinc concentration was 5 Zn/6ins. Including phenol in the SEC eluent two sharp peaks were seen, at insulin dihexamer size and insulin monomer size, and at 4 Zn/6ins more than 95% was at dihexamer size.

All the formulations were mixed with insulin aspart (3 Zn/6ins) at equal concentration and volume and the content of the collected peaks were specifically analysed. At 4 Zn/6ins more than 95% of B29Nε-hexadecandioyl-γ-Glu desB30 insulin was found at very high molecular weight and more than 95% of aspart was found at monomer size. Including phenol in the eluent two peaks were seen, at insulin dihexamer size and insulin monomer size, and at 4 Zn/6ins more than 95% of the long acting insulin was at dihexamer size separated from more than 95% of insulin aspart centered between monomer and hexamer size.

No blunting of the equimolar formulation mix was seen when the long acting analog formulation contained less than 5% of the monomeric form.

Example 5

Euglycaemic Clamp Studies in Pigs

Formulation

B29Nε-hexadecandioyl-γ-Glu desB30 insulin (ins.deriv.) (to 1200 μM) was suspended in water, dissolved and added glycerol 1.6%, and 16 mM phenol and 16 mM m-cresol. Before zinc addition pH was adjusted to 7.5 by sodium hydroxide, and zinc acetate added in smaller portions of max 1 Zn/6ins up to 3 Zn/6ins (ad 1), to 5.62 Zn/6ins (ad II), and to 6 Zn/6ins (III). Sodium chloride was then added to 10 mM followed by adjustment of pH to 7.5 by sodium hydroxide, and adjustment of volume by water.

Insulin aspart (to 600 μM) was suspended in water and added hydrochloric acid to about pH 2.5, zinc acetate to 3 Zn/6ins, glycerol to 1.6%, 16 mM phenol and 16 mM m-cresol to sodium chloride to 10 mM, pH to 7.5 and water to final volume (IV).

Finally insulin aspart (IV) was mixed to B29Nε-hexadecandioyl-γ-Glu desB30 insulin in molar relation of 1:8 and total zinc concentration of 3.38 Zn/6ins.deriv.(I)(lowZnmix) and 6 Zn/6ins.deriv.(II)(highZnmix).

Animal Experiment

Female pigs (N=8, mean body weight 80 kg) were fasted 18 hours prior to the studies. To investigate the effect of mixing insulin aspart and B29Nε-hexadecandioyl-γ-Glu desB30 insulin, each pig received sc in random order either a lowZnmix (I) or a highZnmix (II) or the two analogues (III and IV) administered separately in the same pig. The doses were 0.9 nmol/kg of insulin aspart and 7.2 nmol/kg of B29Nε-hexadecandioyl-γ-Glu desB30 insulin. The pigs were kept euglycaemic at their individual fasting glucose levels by infusion of a 20% glucose solution. Dependent on changes in plasma glucose concentration adjustments of glucose infusion rate were made empirically. Blood samples were collected 0-24 h for specific ELISA plasma analysis of immunoreactive insulin, and the pharmacokinetic profiles are shown in FIG. 13 and FIG. 14.

CONCLUSION

The degree of blunting of both insulin components in the two mix preparations was examined. A marked blunting of both insulin aspart and insulin 454 was seen with low zinc concentration in the mix (3.38 Zn/6ins.deriv.). However, when the zinc concentration was increased to 6 zinc/6ins.deriv., no blunting of the pharmacokinetic profiles was observed. The glucose infusion rate compared well with the sum of the pharmacokinetic profiles of the individual insulin analogues.

Example 6

Citrate as Zinc Buffer

B29Nε-hexadecandioyl-γ-Glu des(B30) insulin (ins.deriv.) (to 600 μM) was suspended in water, dissolved and added glycerol 1.6%, phenol 16 mM, and m-cresol 16 mM. pH was adjusted to 7.5 by sodium hydroxide, and citrate was added in three formulations to 0.6, 1.8 and 6 mM respectively in connection with zinc acetate to 6 zinc/6 insulin deriv. Sodium chloride was then added to 10 mM and sodium phosphate (pH 7.5) to 5 mM followed by adjustment of pH to 7.5 or 7.8 by sodium hydroxide, and adjustment of volume by water.

The formulations were studied after storage 2 week at 37 C compared to storage at 5 C by the SEC method described in example 4 using an eluent with 2 mM phenol. Reference formulations without citrate at 3, 5 and 6 Zn/6ins.deriv. were included.

Miscibility of Insulin Aspart (3Zn/6ins) and prolonged acting insulin derivative 30:70, as measured by collecting fractions from SEC and quantifying by HPLC the presence of fast-acting and prolonged-acting insulins in the high molecular weight fraction (peak1) and in the low molecular weight fraction (peak2). Fraction cut and quantitation is following example 4.

Results:

TABLE 2

Formulation: neutral

dissolution of insulin deriv.

600 μM, glycerol

1.6%, phenol 16 mM, m-

cresol 16 mM, citrate and

zinc addition, sodium

Relative

chloride 10 mM, phosphate

Kav

Relative area %

Kav

area %

5 nM, pH 7.5

SEC eluent:

peak 1

peak1

peak2

peak2

Citrate 0.6 mM, 6

+Phenol

0.54

97

0.73

3

Zn/6ins. deriv., storage

5 C.

Citrate 1.8 mM, 6

+Phenol

0.54

96

0.73

4

Zn/6ins. deriv., storage

5 C.

Citrate 6.0 mM, 6

+Phenol

0.54

96

0.73

4

Zn/6ins. deriv., storage

5 C.

Citrate 0 mM, 3 Zn/6ins.

+Phenol

0.54

94

0.73

6

deriv., storage 5 C.

Citrate 0 mM, 5 Zn/6ins.

+Phenol

0.54

98

0.73

2

deriv., storage 5 C.

Citrate 0 mM, 6 Zn/6ins.

+Phenol

0.54

98

0.73

2

deriv., storage 5 C.

Citrate 0.6 mM, 6

+Phenol

0.54

96

0.73

4

Zn/6ins. deriv., storage 2 w

37 C.

Citrate 1.8 mM, 6

+Phenol

0.54

96

0.73

4

Zn/6ins. deriv., storage 2 w

37 C.

Citrate 6.0 mM, 6

+Phenol

0.54

95

0.73

5

Zn/6ins. deriv., storage 2 w

37 C.

Citrate 0 mM, 3 Zn/6ins.

+Phenol

0.54

93

0.73

7

deriv., storage 2 w 37 C.

Citrate 0 mM, 5 Zn/6ins.

+Phenol

0.54

98

0.73

2

deriv., storage 2 w 37 C.

Citrate 0 mM, 6 Zn/6ins.

+Phenol

0.54

98

0.73

2

deriv., storage 2 w 37 C.

Citrate 0.6 mM, 6

+Phenol

0.54

96

0.73

4

Zn/6ins. deriv., pH 7.8,

storage 2 w 37 C.

Citrate 0.6 mM, 6

+Phenol

0.54

Ins. deriv.

0.73

Ins. aspart

Zn/6ins. deriv., storage

98

97

5 C.

Citrate 1.8 mM, 6

+Phenol

0.54

Ins. deriv.

0.73

Ins. aspart

Zn/6ins. deriv., storage

97

98

5 C.

Citrate 6.0 mM, 6

+Phenol

0.54

Ins. deriv.

0.73

Ins. aspart

Zn/6ins. deriv., storage

97

98

5 C.

Citrate 0 mM, 3 Zn/6ins.

+Phenol

0.54

Ins. deriv.

0.73

Ins. aspart

deriv., storage 5 C.

95

96

Citrate 0 mM, 5 Zn/6ins.

+Phenol

0.54

Ins. deriv.

0.73

Ins. aspart

deriv., storage 5 C.

98

99

Citrate 0 mM, 6 Zn/6ins.

+Phenol

0.54

Ins. deriv.

0.73

Ins. aspart

deriv., storage 5 C.

97

97

Citrate 0.6 mM, 6

+Phenol

0.54

Ins. deriv.

0.73

Ins. aspart

Zn/6ins. deriv., storage

97

96

2 w 37 C.

Citrate 1.8 mM, 6

+Phenol

0.54

Ins. deriv.

0.73

Ins. aspart

Zn/6ins. deriv., storage

97

97

2 w 37 C.

Citrate 6.0 mM, 6

+Phenol

0.54

Ins. deriv.

0.73

Ins. aspart

Zn/6ins. deriv., storage

96

97

2 w 37 C.

Citrate 0 mM, 3 Zn/6ins.

+Phenol

0.54

Ins. deriv.

0.73

Ins. aspart

deriv., storage 2 w 37 C.

93

79

Citrate 0 mM, 5 Zn/6ins.

+Phenol

0.54

Ins. deriv.

0.73

Ins. aspart

deriv., storage 2 w 37 C.

98

97

Citrate 0 mM, 6 Zn/6ins.

+Phenol

0.54

Ins. deriv.

0.73

Ins. aspart

deriv., storage 2 w 37 C.

98

96

CONCLUSION

Using a SEC isotonic saline eluent comprising 2 mM phenol the long acting insulin derivative is predominantly associated at a size of albumin corresponding to a dihexameric form (and not higher associated forms), and the amount of insulin derivative in the monomeric form is decreased at zinc concentration increased from 3 to 5 and 6 Zn/6ins.deriv. Adding 1, 3 or 10 citrate equivalents to the zinc concentration at 6 Zn/6ins.deriv. showed less content of the monomeric insulin derivative compared to a reference at 3 Zn/ins.deriv. The self associating pattern was not changed after storage 2 weeks at 37° C.

Miscibility of Insulin Aspart (3Zn/6ins) formulated without citrate and prolonged acting insulin derivative formulated as shown in the series above in this example—in the molar proportion of 30:70—is shown in the table after storage at 5 C and 2 weeks at 37 C. The insulin derivative formulations comprising citrate at three levels and 6 Zn/6ins were shown to be mixable with insulin aspart after 2 weeks storage at 37 C, whereas insulin aspart was partly included in the high molecular weight fraction at the normal level of zinc at 3 Zn/6ins.

Example 7

Zinc Citrate is Added

B29Nε-hexadecandioyl-γ-Glu desB30 insulin (ins.deriv.) (to 600 μM) is suspended in water, dissolved, (if needed by addition of sodium hydroxide), and added glycerol 1.6%, phenol 16 mM, and m-cresol 16 mM. pH is adjusted to 7.5 by sodium hydroxide, and zinc citrate is added to 0.6 mM Zinc ion. Sodium chloride is then added to 10 mM and sodium phosphate (pH 7.5) to 5 mM followed by adjustment of pH to 7.5 and adjustment of volume by water.

Example 8

A surfactant is Added and Mixtures with Rapid Acting Analogues

B29Nε-hexadecandioyl-γ-Glu desB30 insulin (ins.deriv.) (to 600 μM) is suspended in water, dissolved and added glycerol 1.6%, and 16 mM phenol and 16 mM m-cresol. pH is adjusted to 7.5 by sodium hydroxide, and zinc acetate to 6 zinc/6 insulin deriv. (optionally zinc as citrate). Sodium chloride is then added to 10 mM, a surfactant eg. poloxamer 188 or polysorbate 20 to about 0.002% and sodium phosphate (pH 7.5) to 5 mM followed by adjustment of pH to 7.5 and adjustment of volume by water.

Example 9

Table 3 shows SEC, measured as described in example 4 with 2 or 6 Zn(II) per 6 insulins. For preparation of the compounds mentioned in the table, see WO2006/082204 and WO2006/082205

TABLE 3

Formulation: neutral dissolution of

600 μM insulin, glycerol 1.6%, phenol

16 m, m-cresol 16 mM, 2 or 6

Kav

Relative

Kav

zinc/hexamer, sodium chloride 10 mM,

2 Zn

area %

6 Zn

Relative area %

phosphate 7 mM, pH 7.5

peak 1

peak 1

peak 1

peak 1

B29Nε-(4-{[(2-Carboxy-ethyl)-(15-

0.01

84

0.00

98

carboxy-pentadecanoyl)-amino]-

methyl}-benzoyl) desB30 human

insulin

B29Nε-hexadecandioyl-gamma-Glu-(3-

0.09

79

0.06

89

(2-{2-[2-(2-amino-ethoxy)-ethoxy]-

ethoxy}-ethoxy)-propionyl desB30 human

insulin

B29Nε-(4-{[(2-Carboxy-ethyl)-(14-

0.09

55

0.00

96

carboxy-tetradecanoyl)-amino]-

methyl}-benzoyl) desB30 human insulin

B29Nε-[(5-{[(2-Carboxy-ethyl)-(15-

0.02

81

0.03

96

carboxy-pentadecanoyl)-amino]-

methyl}-furan-2-carbonyl) desB30 human

insulin

B29Nε-hexadecandioyl-gamma-Glu-(4-

0.15

48

0.00

97

aminomethyl-benzoyl) desB30 human

insulin

B29Nε-(2-{[(2-Carboxy-ethyl)-(15-

0.16

54

0.03

93

carboxy-pentadecanoyl)-amino]-

methyl}-benzoyl) desB30 human insulin

Example 10

Preparation of 1,16-hexadecanedioic Acid Mono Benzyl Ester

Hexadecanedioic acid (20.0 g, 69.8 mmol), n-octane and Dowex® are suspended and heated to reflux. Benzyl formate (22.0 g, 162 mmol) is added. After 6 hours additional benzyl formate (22.0 g, 162 mmol) is added. The heating is continued for 50 hours. The reaction mixture is filtered at 80° C. The filtrate is cooled to 20° C., and the precipitate is collected by filtration. The crude product (20.2 g) is suspended in dichloromethane (220 ml) at 20° C. for 4 hours. The suspension is filtered, and the filtrate is evacuated to dryness at 20-30° C. The resulting solid (13.9 g) is recrystallised from 2-propanol (140 ml).

The product is isolated by filtration, and dried to constant weight under reduced pressure at 30-40° C. Yield: 10.2 g (39%) of white material.

1,16-hexadecanedioic acid mono benzyl ester (20.0 g, 53.1 mmol) is dissolved in acetone at 35-40° C. N-Hydroxysuccinimide (6.42 g/55.8 mmol) is added. To the resulting solution dicyclohexylcarbodiimide (DCC) (12.1 g/58.4 mmol) is added. The reaction mixture is stirred for 3-4 hours at 35° C. To the resulting suspension triethylamine (7.40 ml, 53.1 mmol) og L-glutamic acid α-benzyl ester (12.6 g/53.1 mmol) are added. The reaction mixture is stirred for 8-16 hours at 35-40° C. The reaction mixture is cooled to 20-25° C. Methanesulfonic acid (3.45 ml/53.1 mmol) and DCC (12.1 g/53.1 mmol) are added. The reaction mixture is stirred for 8-16 hours at 20-25° C. The reaction mixture is filtered, and the filtrate evacuated to dryness. The residue is partitioned between water (100 ml) and toluene (200 ml). The toluene phase is dried by distilling of water. Silica gel (20 g) is added to the residue. The suspension is stirred for 30 minutes at 20-25° C., then filtered. The volume of the filtrate is reduced to ca 100-120 ml by evaporation under reduced pressure. N-heptane (150 ml) is added over a period of 15-30 minutes. The resulting suspension is stirred for 2 hours. The product is isolated by filtration, and dried to constant weight under reduced pressure at 20-25° C. Yield 21 g (58%) of white material.

L-2-(15-benzyloxycarbonyl-pentadecanoylamino)-pentanedioic acid 5-benzyl ester 1-(2,5-dioxo-pyrrolidin-1-yl) ester (5.0 g, 7.3 mmol) is dissolved in acetone (95 ml) containing trifluoroacetic acid (95 μl). Palladium on carbon, 10% (0.50 g) is added. Under stirring at 30-35° C. hydrogen is added. When the consumption of hydrogen stops the reaction mixture is filtered. The filtrate is cooled to 20° C. and n-heptane (140 ml) is added over a period of 15-30 minutes. The resulting suspension is cooled to 0-5° C. for 2-3 hours. The product is isolated by filtration, and dried to constant weight under reduced pressure at 20-25° C. Yield 3 g (84%) of white material.

The product is analysed by proton NMR (Bruker 600 MHz) using acetone-d6 as solvent.

Acylation of the Human Insulin desB30 ε-aminogroup in Lysine in Position B29 with PC2414

4 g of desB30 human insulin is suspended in 64 g of purified water. 1.85 ml of triethyl amine (TEA) is added to dissolve desB30 human insulin and to raise the pH to 11.4-12.0. The solution is cooled to 2-5° C.

A 150×4.6 mm I.D. column packed with a octyldimethylsilyl substituted silica having pore size of about 100 Å and particle diameter of about 3.5 μm and equilibrated at 40° C. at a flow rate of 1 ml/min with a mixture consisting of 1: a buffer of 20 mM NaH2PO4.H2O and 100 mmol Na2SO4 adjusted to pH 5.9 with NaOH in the aqueous buffer containing 7.8% (w/w) and 2: acetonitrile solvent containing 42.8% w/w acetonitrile, to make 25% (w/w) acetonitrile.

LysB29(Nε-hexadecandioyl-γ-glutamyl) des(B30) human insulin emerged from the column after about 20 min. desB30 human insulin emerged from the column after about 6 min.

Assay (II)

Potency of the Insulin Derivatives of the Invention Relative to Human Insulin

Sprague Dawley male rats weighing 238-383 g on the experimental day were used for the clamp experiment. The rats had free access to feed under controlled ambient conditions and were fasted overnight (from 3 pm) prior to the clamp experiment.

Experimental Protocol

The rats were acclimatized in the animal facilities for at least 1 week prior to the surgical procedure. Approximately 1 week prior to the clamp experiment Tygon catheters were inserted under halothane anaesthesia into the jugular vein (for infusion) and the carotid artery (for blood sampling) and exteriorised and fixed on the back of the neck. The rats were given Streptocilin vet. (Boehringer Ingelheim; 0.15 ml/rat, i.m.) post-surgically and placed in an animal care unit (25° C.) during the recovery period. In order to obtain analgesia, Anorphin (0.06 mg/rat, s.c.) was administered during anaesthesia and Rimadyl (1.5 mg/kg, s.c.) was administered after full recovery from the anaesthesia (2-3 h) and again once daily for 2 days.

The clamp technique employed was adapted from (1). At 7 am on the experimental day overnight fasted (from 3 pm the previous day) rats were weighed and connected to the sampling syringes and infusion system (Harvard 22 Basic pumps, Harvard, and Perfectum Hypodermic glass syringe, Aldrich) and then placed into individual clamp cages where they rested for ca. 45 min before start of experiment. The rats were able to move freely on their usual bedding during the entire experiment and had free access to drinking water. After a 30 min basal period during which plasma glucose levels were measured at 10 min intervals, the insulin derivative to be tested and human insulin (one dose level per rat, n=6-7 per dose level) were infused (i.v.) at a constant rate for 300 min. Plasma glucose levels were measured at 10 min intervals throughout and infusion of 20% aqueous glucose was adjusted accordingly in order to maintain euglyceamia. Samples of re-suspended erythrocytes were pooled from each rat and returned in about ½ ml volumes via the carotid catheter.

On each experimental day, samples of the solutions of the individual insulin derivatives to be tested and the human insulin solution were taken before and at the end of the clamp experiments and the concentrations of the peptides were confirmed by HPLC. Plasma concentrations of rat insulin and C-peptide as well as of the insulin derivative to be tested and human insulin were measured at relevant time points before and at the end of the studies. Rats were killed at the end of experiment using a pentobarbital overdose.

Assay (III)

Determination in Pigs of T50%of the Insulin Derivatives of the Invention

T50%is the time when 50% of an injected amount of the A14 Tyr[125I] labelled derivative of an insulin to be tested has disappeared from the injection site as measured with an external γ-counter.

The principles of laboratory animal care were followed, Specific pathogen-free LYYD, non-diabetic female pigs, cross-breed of Danish Landrace, Yorkshire and Duroc, were used (Holmenlund, Haarloev, Denmark) for pharmacokinetic and pharmacodynamic studies. The pigs were conscious, 4-5 months of age and weighing 70-95 kg. The animals were fasted overnight for 18 h before the experiment.

At the beginning of the experiments a dose of 60 nmol of the insulin derivative according to the invention (test compound) and a dose of 60 nmol of insulin detemir (both 125I labelled in Tyr A14) were injected at two separate sites in the neck of each pig.

The disappearance of the radioactive label from the site of sc. injection was monitored using a modification of the traditional external gamma-counting method (Ribel, U. Subcutaneous absorption of insulin analogues. Berger, M. and Gries, F. A. 70-77 (1993). Stuttgart; New York, Georg Thime Verlag (Conference Proceeding)). With this modified method it was possible to measure continuously the disappearance of radioactivity from a subcutaneous depot for several days using cordless portable device (Scancys Laboratorieteknik, Værløse, DK-3500, Denmark). The measurements were performed at 1-min intervals, and the counted values were corrected for background activity.